Cyclic dinucleotides for cytokine induction

ABSTRACT

wherein X1 is H or F; X2 is H or F; at least one among X1 and X2 is a fluorine atom; Z is OH, OR1, SH or SR1, wherein: R1 is Na or NH4, or R1 is an enzyme-labile group which provides OH or SH in vivo such as pivaloyloxymethyl; B1 and B2 are bases chosen from Adenine, Hypoxanthine or Guanine, and B1 is a different base than B2 and a pharmaceutically acceptable salt thereof. Pharmaceutical compositions including the cyclic dinucleotide, as well as their use in the treatment of a bacterial infection, a viral infection or a cancer are also described.

FIELD OF THE INVENTION

The present invention is in the field of immunotherapy. It concernscyclic dinucleotides (CDNs) of Formulas I, II and III. In particular, itconcerns fluorinated deoxyribo-CDNs and pharmaceutically acceptablesalts and prodrugs thereof, which are able to induce production of TypeI interferons in human and animal cells. The cytokine-induction activityof these cyclic dinucleotides requires the presence of the eukaryoticcellular receptor stimulator of interferon genes (STING), asdemonstrated in vitro.

BACKGROUND OF THE INVENTION Cytokine Induction Immunotherapy

Immunotherapy is a rapidly expanding area of medical treatment in whicha patient's immune system is deliberately activated, suppressed orotherwise modulated for therapeutic benefit. Immunotherapy agentsinclude cells, antigens (e.g. fragments of bacteria or viruses),antibodies, nucleic acids, peptides, proteins, naturally occurringligands and synthetic molecules. Cytokines are small glycoproteinmessengers known chiefly for their role in orchestrating immune responsethrough complex signaling networks, although they also performnon-immune functions. They have been extensively explored asimmunotherapy agents. However, direct administration of cytokines asimmunotherapy is limited by numerous factors, including the very shorthalf-life of cytokines in blood, which must be compensated for withfrequent dosing and high doses. One highly promising immunotherapyapproach is cytokine induction, whereby the patient is treated with animmunomodulatory agent that triggers the production of one or moretherapeutically beneficial cytokines in their body as needed.

STING, Cytokines and Immune Response

A major player in physiological production of cytokines is stimulator ofinterferon genes (STING; also known as ERIS, MITA, MPYS, or TM173), atransmembrane receptor protein that is paramount in innate immunity.Human STING is encoded by the gene TMEM173. Activation of STING leads toproduction of Type I interferons (e.g. IFN-α and IFN-β), via the IRF3(interferon regulatory factor 3) pathway; and to production ofpro-inflammatory cytokines (IL-1α, IL-1β, IL-2, IL-6, TNF-α, etc.), viathe oncogenic transcription factor NF-κB (nuclear factorkappa-light-chain-enhancer of activated B cells) pathway. Moreover,researchers recently reported that in response to viral infection, STINGactivates STAT6 (signal transducer and activator of transcription 6) toinduce (Th2-type), increase (IL-12) or decrease (IL-10) production ofvarious cytokines, including the chemokines CCL2, CCL20, and CCL26 (Chenet al., 2011)

STING Aqonists

Human STING is currently known to be activated three ways: via bindingof exogenous (3′,3) cyclic dinucleotides (c-diGMP, c-diAMP and c-GAMP)that are released by invading bacteria or archaea (see (Gomelsky, 2011)and references therein); via binding of (2′,3′)cyclic guanosinemonophosphate-adenosine monophosphate ((2′,3′)c-GAMP), a recentlydiscovered endogenous cyclic dinucleotide that is produced by the enzymecyclic GMP-AMP synthase (cGAS; also known as C6orf150 or MB21D1) in thepresence of exogenous double-stranded DNA (e.g. that released byinvading bacteria, viruses or protozoa) or of self-DNA in mammals (see,for example: (Ablasser et al., 2013) and (Zhang et al., 2013)); or viabinding of synthetic ligands, such as analogs of the aforementionednaturally-occurring cyclic dinucleotides (see, for example: (Dubensky,Kanne, & Leong, 2013) and (Li et al., 2014)).

Modulation of STING in Immunotherapy

Inspired by the interplay among STING, cytokines and immune response, aswell as by the ever-growing body of knowledge on the clinicalimplications of STING and its mutations, researchers have very recentlybegun to explore STING as a therapeutic target for myriad indications.New STING agonists are being pursued as therapeutic agents for human andanimal health in areas such as cancer or infectious diseases. The knowncyclic dinucleotide STING agonists are an excellent class of compoundson which to base analogs that might exhibit interesting biologicalactivities or desirable drug-like properties. The present inventioncomprises novel cyclic dinucleotides for therapeutic use in human andanimal health.

DETAILED DESCRIPTION OF THE INVENTION CDN STING Aqonists

Some examples of cyclic dinucleotide (CDN) STING agonists are describedin US/2014/0329889 and WO/2014/189805. However, the authors onlychemically synthesized and biologically tested a very small number ofthe compounds that would be theoretically possible from the extremelygeneral chemical structure drawings that they provide. They do notdisclose any detailed structure-activity relationships or describe anyclass effects of particular structural classes of CDNs. Consequently,the authors provide scant rationale to corroborate the choice of onestructural class of CDNs over another in terms of actual STING activityor other properties (e.g. drug-like properties) for a desiredSTING-related application. More recently, WO/2015/077354(PCT/US2014/066436) explored STING agonists, including CDNs, all ofwhich are (2′,3′)-CDNs in which both nucleotides contain a ribose sugarmoiety and the two nucleotides are connected by phosphorothioate diesterlinkages.

Knowledqe Gaps on CDN STING Aqonists

Very little is known about the biological activity of differentstructural classes of CDNs. Specifically, there is scant patentprecedent on class effects according to CDN structural class, nor isthere any patent precedent on how such class effects might bespecifically exploited for specific therapeutic, diagnostic or researchapplications related to STING activity. Such information will becritical for discovery and exploitation of novel CDN STING agonists withdesirable properties for therapeutic, diagnostic or researchapplications based on manipulation of STING activity.

The Present Invention

Considering the paucity of reported structure-activity relationships forSTING agonists, the field of STING agonists is ripe for invention. Inthis context, the present invention concerns fluorinated deoxyribo-CDNSTING agonists that exhibit unique, unobvious and previously unreportedclass effects relative to their corresponding non-fluorinated analogs.

In our work exploring cyclic dinucleotides (CDNs) as immunomodulatorycompounds and potential agonists of STING, we initially sought tosynthesize and assay CDNs of the following Formulas (I), (II) and (III):

wherein:

-   -   X₁ is H, OH, or F;    -   X₂ is H, OH, or F;    -   Z is OH, OR₁, SH or SR₁, wherein:        -   i) R₁ is Na or NH₄, or        -   ii) R₁ is an enzyme-labile group which provides OH or SH in            vivo such as pivaloyloxymethyl;    -   B₁ and B₂ are bases chosen from:

with the proviso that:

-   -   in Formula (I): X₁ and X₂ are not OH,    -   in Formula (II): when X₁ and X₂ are OH, B₁ is not Adenine and B₂        is not Guanine, and    -   in Formula (III): when X₁ and X₂ are OH, B₁ is not Adenine, B₂        is not Guanine and Z is not OH.

In the present invention, the term “cyclic dinucleotide” (abbreviated as“CDN”) represent a class of cyclic molecules with two phosphodiesterlinkages, or two phosphorothioate diester linkages, or onephosphodiester linkage and one phosphorothioate diester linkage, betweentwo nucleosides. This includes (3′,5′)-(3′,5′) nucleotide linkages(abbreviated as (3′,3′)); (3′,5′)-(2′,5′) nucleotide linkages(abbreviated as (3′,2′)); (2′,5′)-(3′,5′) nucleotide linkages(abbreviated as (2′,3′)); and (2′,5′)-(2′,5′) nucleotide linkages(abbreviated as (2′,2′)).

The term “nucleoside” refers to a glycosylamine comprising a nitrogenousbase and a five-carbon sugar, wherein the nitrogenous base is bound tothe five-carbon sugar via a beta-glycosidic linkage.

The term “nucleotide” refers to any nucleoside linked to a phosphategroup at position 5′, 3′ or 2′ of the sugar moiety.

“Pharmaceutically acceptable salts” include those derived frompharmaceutically acceptable inorganic or organic bases and acids.Suitable salts include those derived from alkali metals such aspotassium and sodium, alkaline earth metals such as calcium andmagnesium, among numerous other acids well known in the pharmaceuticalart.

The term “pharmaceutically acceptable prodrug” herein refers to acompound that is metabolized, for example hydrolyzed or oxidized, in thehost (i.e. the human or animal subject that receives the compound) toform the compound of the present invention. Typical examples of prodrugsinclude compounds that have biologically labile protecting groups onfunctional moieties of the active compound. Prodrugs include compoundsthat can be oxidized, reduced, aminated, deaminated, hydroxylated,dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated,acylated, deacylated, phosphorylated or dephosphorylated to produce theactive compound.

The term “prodrug”, as used herein, relates to an inactive or activederivative of a compound represented by the structural Formula (I), (II)or (III) as defined herein above or any one of their specificembodiments, which undergoes spontaneous or enzymatic transformationwithin the body of an animal, e.g. a mammal such as a human being, inorder to release the pharmacologically active form of the compound. Fora comprehensive review, see (Rautio et al., 2008).

In particular for the purpose of the present invention, prodrugs of thecompounds represented by the structural Formula (I), (II) or (III)including any one of the above-described specific embodiments thereof,may be formed as described in detail in (Hecker & Erion, 2008).

Phosphate prodrug can take the form of an ester, in particularacyloxyalkyl esters (e.g. pivaloyloxymethyl ester (POM)) orS-acylthioethyl (SATE) esters, a carbonate, a carbamate or an amide,such as amino acid prodrugs.

The expression “enzyme-labile protecting group” denotes a group designedto enable passive diffusion of a compound across cellular or parasiticmembranes via charge-masking, such that, once inside the cell orparasite interior, the compound undergoes an enzymatic transformation(or deprotection) that affords an OH or SH group. Examples ofenzyme-labile protecting groups include for instance acyloxyalkyl groupssuch as pivaloyloxymethyl (POM) or S-acylthioethyl (SATE) or amino acidgroups.

In the present description, it is considered that the expression “cyclicdinucleotides of Formula (I), (II) or (III)” is considered to alsoinclude the pharmaceutically acceptable salts or a pharmaceuticallyacceptable prodrugs of said cyclic dinucleotides of Formula (I), (II) or(III).

A particular class of cyclic dinucleotides of Formula (I), (II) or (III)is when B₁ or B₂ is Guanine.

A particular class of cyclic dinucleotides of Formula (I), (II) or (III)is when B₁ or B₂ is Adenine.

A particular class of cyclic dinucleotides of Formula (I), (II) or (III)is when B₁ or B₂ are Hypoxanthine.

A particular class of cyclic dinucleotides of Formula (I), (II) or (III)is when B₁ and B₂ are independently chosen from Guanine or Adenine.

A particular class of cyclic dinucleotides of Formula (I), (II) or (III)is when B₁ or B₂ are independently chosen from Guanine or Hypoxanthine.

A particular class of cyclic dinucleotides of Formula (I), (II) or (III)is when B₁ and B₂ are independently chosen from Adenine or Hypoxanthine.

A particular class of cyclic dinucleotides of Formula (I), (II) or (III)is when Z is OH.

A particular class of cyclic dinucleotides of Formula (I), (II) or (III)is when Z is SH.

A particular class of cyclic dinucleotides of Formula (I), (II) or (III)is when Z is OH or SH.

A particular class of cyclic dinucleotides of Formula (I), (II) or (III)is when Z is SR₁, wherein:

-   -   i) R₁ is Na or NH₄, or    -   ii) R₁ is an enzyme-labile group which provides OH or SH in vivo        such as pivaloyloxymethyl.

A particular class of cyclic dinucleotides of Formula (I), (II) or (III)is when B₁ and B₂ are independently chosen from Adenine or Hypoxanthineand X₁ and X₂ are identical. Preferably, X₁ and X₂ are OH or a fluorineatom. In one particular embodiment, Z is OH or SH.

A particular class of cyclic dinucleotides of Formula (I), (II) or (III)is when B₁ and B₂ are independently chosen from Guanine or Hypoxanthineand X₁ and X₂ are identical. Preferably, X₁ and X₂ are OH or a fluorineatom. In one particular embodiment, Z is OH or SH.

A particular class of cyclic dinucleotides of Formula (I), (II) or (III)is when B₁ and B₂ are independently chosen from Adenine or Guanine andX₁ and X₂ are identical. Preferably, X₁ and X₂ are OH or a fluorineatom. In one particular embodiment, Z is OH or SH.

A particular class of cyclic dinucleotides of Formula (I), (II) or (III)is when X₁ and X₂ are not a fluorine atom.

A particular class of cyclic dinucleotides of Formula (I), (II) or (III)is when at least one among X₁ and X₂ is a fluorine atom. In oneparticular embodiment, X₁ and X₂ are both a fluorine atom.

A particular class of cyclic dinucleotides of Formula (I), (II) or (III)is the following cyclic dinucleotides (each of which is provided with afive-character code of the format “C L###”):

or a pharmaceutically acceptable salt or prodrug thereof.

A particular class of cyclic dinucleotides of Formula (I), (II) or (III)is when one or both nucleosides are modified at the 2′ position. Thisclass comprises the following compounds:

We identified a structurally unprecedented subset of these CDNs thatshowed surprising biological activity and that have never previouslybeen reported as STING agonists. These CDNs form the basis for thepresent invention and are distinct from previously reported CDN STINGagonists (see, for example: US/2014/0329889 and WO/2014/189805). Thus,in one aspect, the present invention provides CDNs that are defined byall of the following structural criteria: firstly, unlike in naturallyoccurring CDNs, in which the sugar moiety of each nucleotide is aribose, in the CDNs of the present invention the sugar moiety of eachnucleotide is a 2′-deoxyribose; secondly, in the CDNs of the presentinvention, either the 2′ position of the sugar moiety in bothnucleotides must be substituted with a fluorine atom, or the 2′ positionof the sugar moiety of one nucleotide must be substituted with afluorine atom while the 2′ position of the sugar moiety of the othernucleotide must be substituted with a hydrogen atom; thirdly, in theCDNs of the present invention, the base in each nucleotide is preferablychosen from guanine, adenine or hypoxanthine, with the proviso that thetwo nucleotides of the CDN cannot contain the same base.

Thus, in one embodiment, the present invention provides CDNs of Formula(I) according to Claim 1, i.e. compounds of Formula (I):

wherein:

-   -   X₁ is H or F;    -   X₂ is H or F;    -   At least one among X₁ and X₂ is a fluorine atom;    -   Z is OH, OR₁, SH or SR₁, wherein:        -   i) R₁ is Na or NH₄, or        -   ii) R₁ is an enzyme-labile group which provides OH or SH in            vivo such as pivaloyloxymethyl;    -   B₁ and B₂ are bases chosen from:

and B₁ is a different base than B₂,or a pharmaceutically acceptable salt thereof.

The expression “B₁ is a different base than B₂” includes B₁ is Guanineand B₂ is Adenine or B₁ is Guanine and B₂ is Hypoxanthine or B₁ isAdenine and B₂ is Guanine or B₁ is Adenine and B₂ is Hypoxanthine or B₁is Hypoxanthine and B₂ is Guanine or B₁ is Hypoxanthine and B₂ isAdenine.

A particular class of cyclic dinucleotides of Formula (I) as definedabove is when B₁ and B₂ are different and both X₁ and X₂ are F.

A particular class of cyclic dinucleotides of Formula (I) as definedabove is when B₁ and B₂ are different, both X₁ and X₂ are F and Z is OH.

A particular class of cyclic dinucleotides of Formula (I) as definedabove is when B₁ and B₂ are different, both X₁ and X₂ are F and Z is OR₁as defined above.

A particular class of cyclic dinucleotides of Formula (I) as definedabove is when B₁ and B₂ are different, both X₁ and X₂ are F and Z is SHas defined above.

A particular class of cyclic dinucleotides of Formula (I) as definedabove is when B₁ and B₂ are different, both X₁ and X₂ are F and Z is SR₁as defined above.

A particular class of cyclic dinucleotides of Formula (I) as definedabove is when B₁ and B₂ are different and X₁ and X₂ are different (i.e.X₁═H and X₂═F or X₁═F and X₁═H).

A particular class of cyclic dinucleotides of Formula (I) as definedabove is when B₁ and B₂ are different, X₁ and X₂ are different and Z isOH.

A particular class of cyclic dinucleotides of Formula (I) as definedabove is when B₁ and B₂ are different, X₁ and X₂ are different and Z isOR₁ as defined above.

A particular class of cyclic dinucleotides of Formula (I) as definedabove is when B₁ and B₂ are different, X₁ and X₂ are different and Z isSH as defined above.

A particular class of cyclic dinucleotides of Formula (I) as definedabove is when B₁ and B₂ are different, X₁ and X₂ are different and Z isSR₁ as defined above.

The cyclic dinucleotides of the present invention induce Type Iinterferons and/or pro-inflammatory cytokines in vitro in human cells,animal cells and human blood. The cytokine-induction activity of thesecyclic dinucleotides requires the presence of STING, as confirmed by invitro experiments in human or animal cells.

The cyclic dinucleotides of the invention are agonists of the receptorSTING.

The term “agonist” refers to any substance that activates a biologicreceptor in vitro or in vivo to provoke a physiologic response.

“STING” is an abbreviation of “stimulator of interferon genes”, which isalso known as “endoplasmic reticulum interferon stimulator (ERIS)”,“mediator of IRF3 activation (MITA)”, “MPYS” or “transmembrane protein173 (TM173)”. STING is a transmembrane receptor protein that in humansis encoded by the gene TMEM173. Activation of STING by cyclicdinucleotides (CDN) leads to activation of the IRF3 and NF-κB pathwaysand consequently, to induction of Type I interferons and ofpro-inflammatory cytokines, respectively. In response to viralinfection, STING activates STAT6 (signal transducer and activator oftranscription 6) to induce (Th2-type), increase (IL-12) or decrease(IL-10) production of various cytokines, including the chemokines CCL2,CCL20, and CCL26 (Chen et al., 2011).

The term “STING agonist” herein refers to a substance that activates thereceptor STING in vitro or in vivo. According to the invention, acompound is deemed to be a STING agonist if:

-   -   it induces Type I interferons in vitro in human or animal cells        that contain active STING; and    -   it does not induce Type I interferons in vitro in human or        animal cells that do not contain active STING.

A typical test to ascertain whether a ligand is a STING agonist is toincubate the ligand in a wild-type human or animal cell line and in thecorresponding cell line in which the STING coding gene has beengenetically inactivated by small or long base deletions (e.g. ahomozygous STING knockout cell line). An agonist of STING will induceType I interferons in the wild-type cells but will not induce Type Iinterferons in the cells in which the STING coding gene has beeninactivated.

The cyclic dinucleotides of the invention induce Type I interferons invitro in human or animal cells that contain active STING. However, theydo not induce Type I interferons in vitro in human or animal cells thatdo not contain active STING.

The present invention is concerned with fluorinated deoxyribo-cyclicdinucleotides (CDNs). Specifically, it is concerned with(3′,3′)-2′(mono- or di-fluorinated)-2′-deoxyribo-(CDNs).

The cyclic dinucleotides of Formula (I), (II) or (III) wherein at leastone among X₁ and X₂ is a fluorine atom, in particular X₁ and X₂ are botha fluorine atom, and Z is OH induce more Type I interferon and NF-κBactivity than do their non-fluorinated counterparts in human and murinecell lines.

The cyclic dinucleotides of Formula (I), (II) and (III) wherein at leastone among X₁ and X₂ is a fluorine, in particular X₁ and X₂ are both afluorine atom, and Z is OH or SH exhibit slower elimination from bloodafter intravenous injection in mice, i.e. longer kinetics of clearance,and exhibit greater resistance to enzymatic cleavage in vitro; ascompared to their corresponding non-fluorinated CDNs.

Salts or Prodrug Formulations of the Cyclic Dinucleotides

Pharmaceutically acceptable salts include those derived frompharmaceutically acceptable inorganic or organic bases and acids.Suitable salts include those derived from alkali metals such aspotassium and sodium, alkaline earth metals such as calcium andmagnesium, among numerous other acids well known in the pharmaceuticalart. The term “pharmaceutically acceptable prodrugs” refers to acompound that is metabolized, for example hydrolyzed or oxidized, in thehost (i.e. the human or animal subject that receives the compound) toform the compound of the present invention. Typical examples of prodrugsinclude compounds that have biologically labile protecting groups onfunctional moieties of the active compound. Prodrugs include compoundsthat can be oxidized, reduced, aminated, deaminated, hydroxylated,dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated,acylated, deacylated, phosphorylated or dephosphorylated to produce theactive compound.

The CDN prodrugs described herein can be administered to additionallyincrease the activity, bioavailability or stability, or otherwise alterthe properties of the CDN monophosphate.

A number of CDN prodrug ligands are known. In general, alkylation,acylation or other lipophilic modifications on the phosphate moiety, orthe use of other analog of the nucleoside, will increase the stabilityof the nucleotide.

Examples of substituent groups that can replace one or more hydrogens onthe phosphate moiety are alkyl, aryl, steroids, carbohydrates,including, but not limited to, sugars, 1,2-diacylglycerol and alcohols.Many are described in (Jones, 1995).

Use of the Compounds of the Invention

Another object of the present invention is the cyclic dinucleotides ofFormula (I), (II) or (Ill) for use in a therapeutic treatment in humansor animals.

In particular, the compounds of the present invention may be used fortherapeutic or diagnostic applications in human or animal health. Thefollowing examples serve to illustrate different possible applicationsof the present invention; however, these examples are not intended tolimit the scope of the invention.

The term “therapeutic agent” refers to one or more substances that areadministered to a human or animal in order to achieve some kind oftherapeutic effect in that human or animal, including to prevent, cure,or mitigate the effects of, infection or disease, and/or to otherwiseimprove the health of that human or animal.

The term “monotherapy” refers to the use of a single substance and/orstrategy to treat a human or animal in any clinical or medical context,as opposed to the use of multiple substances and/or strategies to treata human or animal in the same clinical or medical context, regardless ofwhether the multiple substances and/or strategies are used sequentiallyin any order or concurrently.

The term “chemotherapeutic agent” herein refers to one or more chemicalsubstances that are administered to a human or animal in order to killtumors, or slow or stop the growth of tumors, and/or slow or stop thedivision of cancerous cells and/or prevent or slow metastasis.Chemotherapeutic agents are often administered to treat cancer, but arealso indicated for other diseases.

The term “chemotherapy” refers to medical treatment of a human or animalwith one or more chemotherapeutic agents (see definition above).

The term “chemoimmunotherapy” refers to the combined use, whethersequentially in any order or concurrently, of chemotherapy substancesand/or strategies, and immunotherapy substances and/or strategies.Chemoimmunotherapy is often employed to treat cancer, but can also beemployed to treat other diseases.

The term “immune system” refers to the ensemble, or to any one or morecomponents, of the molecules, substances (e.g. bodily fluids), anatomicstructures (e.g. cells, tissue and organs) and physiologic processesinvolved in preventing infection in the body, in protecting the bodyduring infection or during disease, and/or in helping the body torecuperate after infection or disease. A complete definition of “immunesystem” is beyond the scope of this patent; however, this term should beunderstood by any ordinary practitioner in the field.

The term “immune agent” refers to any endogenous or exogenous substancethat can interact with any one or more components of the immune system.The term “immune agent” includes antibodies, antigens, vaccines andtheir constituent components, nucleic acids, synthetic drugs, natural orsynthetic organic compounds, cytokines, natural or modified cells,synthetic analogs thereof, and/or fragments thereof.

The term “immunotherapy” refers to any medical treatment in which one ormore components of a human's or animal's immune system is deliberatelymodulated in order to directly or indirectly achieve some therapeuticbenefit, including systemic and/or local effects, and preventativeand/or curative effects. Immunotherapy can involve administering one ormore immune agents (see definition above), either alone or in anycombination, to a human or animal subject by any route (e.g. orally,intravenously, dermally, by injection, by inhalation, etc.), whethersystemically, locally or both. “Immunotherapy” can involve provoking,increasing, decreasing, halting, preventing, blocking or otherwisemodulating the production of cytokines, and/or activating ordeactivating cytokines or immune cells, and/or modulating the levels ofimmune cells, and/or delivering one or more therapeutic or diagnosticsubstances to a particular location in the body or to a particular typeof cell or tissue, and/or destroying particular cells or tissue.Immunotherapy can be used to achieve local effects, systemic effects ora combination of both.

The term “immunosuppressed” describes the state of any human or animalsubject whose immune system is functionally diminished, deactivated orotherwise compromised, or in whom one or more immune components isfunctionally diminished, deactivated or otherwise compromised.“Immunosuppression” can be the cause, consequence or byproduct ofdisease, infection, exhaustion, malnutrition, medical treatment or someother physiologic or clinical state.

The terms “immunomodulating substance”, “immunomodulatory substance”,“immunomodulatory agent” and “immunomodulator”, used here synonymously,refer to any substance that, upon administration to a human or animal,directly influences the functioning of the immune system of that humanor animal. Examples of common immunomodulators include, but are notlimited to, antigens, antibodies and small-molecule drugs.

The term “vaccine” refers to a biological preparation administered to ahuman or animal in order to elicit or enhance a specific immune systemresponse and/or protection against one or more antigens in that human oranimal.

The term “vaccination” refers to treatment of a human or animal with avaccine or to the act of administering a vaccine to a human or animal.

The term “adjuvant” refers to a secondary therapeutic substance that isadministered together (either sequentially in any order, orconcurrently) with a primary therapeutic substance to achieve some kindof complimentary, synergic or otherwise beneficial effect that could notbe achieved through use of the primary therapeutic substance alone. Anadjuvant can be used together with a vaccine, chemotherapy, or someother therapeutic substance. Adjuvants can enhance the efficacy of theprimary therapeutic substance, reduce the toxicity or side effects ofthe primary therapeutic substance, or provide some kind of protection tothe subject that receives the primary therapeutic substance, such as,but not limited to, improved functioning of the immune system.

In one embodiment, the cyclic dinucleotide of Formula (I), (II) or (III)can be administered as immunotherapy to a human or an animal to inducein vivo production of one or more cytokines that are therapeuticallybeneficial to that human or animal. This type of immunotherapy could beused alone or in combination with other treatment strategies, whethersequentially in any order, or concurrently. It could be used to prevent,cure, and/or mitigate the effects of, infection or disease in that humanor animal, and/or to modulate the immune system of that human or animalto achieve some other therapeutic benefit.

In one particular embodiment, the cyclic dinucleotides of the presentinvention can be used for cytokine induction immunotherapy ofimmunosuppressed individuals.

In this example, a cyclic dinucleotide of Formula (I) (II) or (III)would be administered to an immunosuppressed human or animal subject toinduce in vivo production of one or more cytokines that directly orindirectly enhance the immune system of that human or animal. Subjectsthat might benefit from such treatment include those suffering fromautoimmune disorders, immune system deficiencies or defects, microbialor viral infections, infectious diseases, or cancer.

The present invention thus discloses a method for inducing cytokine inimmunosuppressed individuals, said method comprising administering to apatient in need thereof a cyclic dinucleotide of Formula (I), (II) or(III) or a pharmaceutically acceptable salt or prodrug thereof.

In another embodiment, the cyclic dinucleotides of the present inventioncan be used for cytokine induction immunotherapy in combination withchemotherapy

In this example, a cyclic dinucleotide of Formula (I) (II) or (III)would be administered together with one or more chemotherapeutic agents,sequentially in any order or concomitantly, to a cancer patient to stopthe growth of, shrink and/or destroy tumors in that patient. Thechemoimmunotherapy resulting from the combination of cytokine induction,provided by the compound(s) of the present invention, and cytotoxicity,provided by the chemotherapeutic agent(s), might be less toxic to thepatient, cause fewer side effects in the patient and/or exhibit greateranti-tumor efficacy than would the chemotherapeutic agent(s) when usedas monotherapy.

The present invention thus discloses a method for treating cancer, saidmethod comprising administering to a patient in need thereof:

-   -   a chemotherapeutic agent; and    -   a cyclic dinucleotide of Formula (I), (II) or (III) or a        pharmaceutically acceptable salt or prodrug thereof.

In another embodiment, the cyclic dinucleotides of the present inventioncan be used for cytokine induction immunotherapy as vaccine adjuvanttherapy.

In this example, a cyclic dinucleotide of Formula (I) (II) or (III)would be administered to a human or animal subject that has received, isreceiving or will receive a vaccination. The benefits provided by thepresent invention might include enhanced efficacy of the vaccinationagainst the target antigen, reduced toxicity of the vaccination, reducedadverse side effects of the vaccination, or enhanced immune protectionof the human or animal subject.

Another object of the present invention is the cyclic dinucleotides ofFormula (I), (II) or (Ill) for use in the treatment of a bacterialinfection, a viral infection or a cancer.

As used herein, “cancer” refers to the physiological condition insubjects that is characterized by unregulated or dysregulated cellgrowth or death. The term “cancer” includes solid tumors and blood-borntumors, whether malignant or benign.

In a preferred embodiment, the cancer is from the following group:bladder cancer, breast cancer, cholangiocellular cancer, leukemia, lungcancer, lymphoma, nasopharyngeal cancer, ovarian cancer, pancreaticcancer and urothelial cancer.

In one particular embodiment, the cancer is a solid pancreatic tumor.

The present invention thus discloses a method for treating a bacterialinfection, a viral infection or a cancer, said method comprisingadministering to a patient in need thereof a cyclic dinucleotide ofFormula (I), (III) or (III) or a pharmaceutically acceptable salt orprodrug thereof.

Another object of the present invention is the cyclic dinucleotides ofFormula (I), (II) or (III) for use in the treatment of a pathology thatmay be alleviated by the induction of an immune response via the STINGpathway.

Another object of the present invention is a kit-of-parts comprising acyclic dinucleotide of Formula (I), (II) or (III) and a chemotherapeuticagent for use in the treatment of solid pancreatic tumors.

The term “kit-of-parts” herein refers to a combined preparation whereinthe active ingredients are physically separated for use in a combinedtherapy by simultaneous administration or sequential administration tothe patient.

Hence, according to the present invention, the chemotherapeutic agentand the cyclic dinucleotide or a pharmaceutically acceptable salt orprodrug thereof are administered to the patient in a separate form,either simultaneously, separately or sequentially in any order, for thetreatment of cancer.

In one embodiment, said chemotherapeutic agent is gemcitabine.

Deoxynucleosides for the Synthesis of Cyclic Dinucleotides

Natural nucleosides, 2′-deoxy-Adenosine (dA), 2′-deoxy-Guanosine (dG),or 2′-deoxy-Inosine (dI) can be used for the synthesis of CDNs of theFormulas (I) and (II).

The 3′-deoxy-Adenine, 3′-deoxy-Guanosine, or 3′-deoxy-Inosine for CDNsof the Formula (II) and (III) refers to a nucleoside unit having a sugarmoiety, for example a ribosyl moiety or a xylosyl moiety that ismodified at the 3′ position such that the hydroxyl group (3′-OH) isreplaced by an hydrogen group.

Fluoro-Substituted Nucleosides for the Synthesis of CDNs

Since STING is located in the endoplasmic reticulum and detects cyclicdinucleotides in the cytoplasm, any STING agonist destined fortherapeutic use must be able to penetrate into cells. Furthermore,greater cellular uptake of a compound translates to higherbioavailability, which is a desirable property for clinical use. In thepresent invention, the fluorinated compounds were designed to explorethe possibility that greater cellular uptake conferred by one or twofluorine atoms would lead to greater Type I interferon inductionactivity than that of the reference compound, c-AIMP, which does notcontain any fluorine atoms.

In the present invention, we surprisingly found that the subject CDNs,all of which contain at least one fluorine atom, are more active asSTING agonists than are their corresponding non-fluorinated analogs.Specifically, the CDNs of the present invention exhibit greaterSTING-dependent cytokine induction activity in human cells, animal cellsand human blood than do their corresponding non-fluorinated analogs

The more quickly a drug is enzymatically degraded in the body, theshorter will be its half-life and consequently, the lesser will be itsactivity. Thus, a desirable property for compounds intended fortherapeutic use is resistance to enzymatic degradation. There are invitro enzymatic cleavage tests that can provide some indication of theresistance of a given compound to common enzymes that degrade compoundsthat are structurally similar to the one being tested. In our work, wewere very surprised to discover yet another distinguishingcharacteristic of the subject CDNs of the present invention: thesefluorinated deoxyribo-CDNs consistently showed superior resistance tocleavage by snake-venom phosphodiesterase (SVPD) or nuclease P1 (NP1),compared to their corresponding non-fluorinated ribo-CDNs.

Monofluoro-Nucleoside

The 2′-deoxy-2′-fluoro of Adenosine, Guanosine or Inosine for CDNs ofFormula (I) and (II) refers to a nucleoside with a modification at the2′ position, such that, the hydroxyl group (2′-OH) is replaced by afluoro group (2′-F).

The 2′-fluoronucleoside derivatives for the synthesis of CDNs of Formula(I) and (II) may be prepared by any of the methods known in the art(see, for example: (Herdewijna, 1989; Thomas, 1994) and (Ross, 1997)).

The 3′-deoxy-3′-fluoro of Adenosine, Guanosine or Inosine for CDN ofFormula (II) and (III) refers to a nucleoside with a modification at the3′ position, such that the hydroxyl group (3′-OH) is replaced by afluoro group (3′-F).

Phosphorothioate Internucleotide Linkage

The phosphorothioate internucleotide linkage refers to the replacementof a P═O group with a P═S group, and includes phosphorodithioateinternucleoside linkages. One or both of the internucleotide linkagesthat are present in the cyclic dinucleotides can be phosphorothioateinternucleotide linkages.

The phosphorous atom in a phosphodiester linkage of CDNs can bedescribed as being “pro-chiral.” Once a non-bonding oxygen atom of thephosphodiester linkage is replaced or modified, a chiral sugar-phosphatelinkage is generated. The resulting intersugar linkage is either an Spintersugar linkage or an Rp intersugar linkage. Replacement of anon-bonding oxygen atom in the natural phosphodiester linkage, with asulfur atom to obtain a phosphorothioate linkage generates a chiralcenter and consequently, affords Sp and Rp diastereomers. Moleculeswherein substantially all of the phosphorous atoms in the sugar backboneare either Sp or Rp are referred to herein as “chirally pure”.

Cyclic dinucleotides are enzymatically degraded by nucleases and/orphosphodiesterases (see, for example: (Li et al., 2014) (Diner et al.,2013) (Danilchanka & Mekalanos, 2013) (Shanahan, Gaffney, Jones, &Strobel, 2013) (Simm, Morr, Kader, Nimtz, & Romling, 2004)) andtherefore, when used as therapeutic agents, these compounds can sufferfrom diminished half-life. The compounds CL655 and CL656 were chosen toenable maximal half-life, and possibly higher activity, in vivo, as theycontain phosphorothioate (also known as “P(S)” or “thiophosphate”)internucleotide linkages. The use of such linkages is a known strategyto circumvent enzymatic hydrolysis (see, for example: US 2014/0205653A1). An example of a phosphorothioate compound that is more resistant toenzymatic hydrolysis than its phosphodiester analog is(2′,3′)c-G^(s)A^(s)MP or (2′,3′) c-GAMP(S) (InvivoGen catalog code:tlrl-scga; Li, 2014). The phosphorothioate linkage introduces anadditional chiral center on the phosphorus atom, which yields adiastereoisomer pair ([Rp] and [Sp]) at each phosphorothioate linkage.In the present invention, CL655 and CL656 were obtained and tested asracemic mixtures.

General Schemes for Preparing Active Compounds

Methods for the easy preparation of cyclic dinucleotides or prodrugsthereof disclosed herein can be prepared as described in detail below,or by other methods known to those skilled in the art. It will beunderstood by one of ordinary skill in the art that these schemes are inno way limiting and that variations of detail can be made withoutdeparting from the spirit and scope of the present invention.

The term “protecting group” as used herein, and unless otherwisedefined, refers to a chemical functional group that is attached to anoxygen, nitrogen or phosphorus atom to prevent further reaction of thatatom, or for other purposes. A wide variety of protecting groups foroxygen and for nitrogen are known to those skilled in the art of organicsynthesis, and are described, for example, in (Wuts, Greene, & Greene,2014).

Generally, cyclic dinucleotides or prodrugs thereof of the Formulas (I),(II) or (III) are prepared by first preparing the correspondingnucleoside, then preparing the 5′-hydroxy group, the functional group(OH) at the 2′ position or 3′ position, and then, if necessary,protecting the exocyclic amine of the purine base. Then, theappropriately protected nucleosides are converted to the corresponding3′-phosphoramidite, 2′-phosphoramidite, 3′-H-phosphonate or2′-H-phosphonate, which constitute the starting material for thepreparation of the cyclic dinucleotides described herein.

The reaction schemes shown below apply to the synthesis of CDNs ofFormula I and can be employed for the synthesis of CDNs of Formulas (II)and (III):

CDNs of Formulas (I) and (II) are prepared by first preparingappropriately protected phosphoramidite of Formula (I_(A)) orH-phosphonate of Formula (I_(F)):

Phosphoramidites of the Formula (I_(A)) and H-phosphonates of theFormula (I_(F)) can be synthesized by first preparing the appropriatelyprotected nucleosides of the Formula (IV), which can be accomplished byone of ordinary skill in the art. Firstly, the base, which has anexocyclic amine, is protected, and then the 3′- and 5′-hydroxyl groupsare simultaneously or selectively protected. The substitutions X₂ areeventually protected, using protecting groups such as TBDMS ortetrahydropyran (Thp) when X₂ is hydroxyl. Finally, the protectinggroups at the 3′- and 5′-hydroxyls are cleaved, and the 5′-hydroxyl issubsequently protected with a trityl derivative to give the nucleosideof the Formula (IV):

Nucleosides of the Formula (IV_(A)) can be prepared by methods outlinedin: (Chu, 2002; Rajagopalan, 2003; Schinazi, 2004; Vorbrüggen, 2001).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. STING signaling in the cell. Activation of STING by cyclicdinucleotides (CDN) leads to activation of the IRF3 and NF-κB pathwaysand consequently, to induction of Type I interferons and ofpro-inflammatory cytokines, respectively.

FIG. 2. In vitro Type I interferon induction activity in THP1-Dual™ cellcultures: non-fluorinated vs. fluorinated cyclic dinucleotides.

FIG. 3. In vitro NF-κB pathway induction in THP1-Dual™ cell cultures:non-fluorinated vs. fluorinated cyclic dinucleotides.

FIG. 4. In vitro Type I interferon induction activity in wild-type vs.STING knockout B16 cells. It shows relative ISG54 activity (as anindirect measurement of Type I interferon induction) of non-fluorinatedvs. fluorinated cyclic dinucleotides incubated in cultures of wild-type(left-side of graph) or STING-knockout (right-side of graph) B16 cellsfor 24 h. WT: wild-type; SKO: STING knockout (homozygous).

FIG. 5. In vitro Type I interferon induction activity in wild-type vs.STING-knockout RAW cells. It shows relative ISG54 activity (as anindirect measurement of Type I interferon induction) of non-fluorinatedvs. fluorinated cyclic dinucleotides incubated in cultures of wild-type(left-side of graph) or STING-knockout (right-side of graph) RAW cellsfor 24 h. WT: wild-type; SKO: STING knockout (homozygous).

FIG. 6. Type I interferon induction activity of cyclic dinucleotides inmice. Measurement of Type I interferon induction in sera from mice at 4h post-treatment.

FIG. 7. IL-6 induction activity of cyclic dinucleotides in mice.Measurement of IL-6 induction in sera from mice at 4 h post-treatment.

FIG. 8. In vivo elimination of cyclic dinucleotides in mice. Temporalchange in plasma concentration of cyclic dinucleotides in treated mice.

FIG. 9. Cytokine induction activity of CDNs in human whole blood.

FIGS. 10 A-D. Resistance of distinct CDNs to cleavage by the enzymesSVPD and NP1 over time, as monitored by HPLC. Compare CL614 and CL656 toc-AIMP, for resistance to SVPD (A) or NP1 (B). Compare CL603 and CL656to c-GAMP, for resistance to SVPD (C) or NP1 (D).

FIG. 11. In vitro activity of distinct CDNs in THP-1 Dual™ cells beforeand after incubation with the enzymes SVPD and NP1.

The invention will be illustrated by the non-limiting followingexamples.

EXAMPLES

Specific compounds that are representative of this invention wereprepared as per the following examples and are offered by way ofillustration to aid in the understanding of the invention. They shouldnot be construed to limit in any way the invention set forth in theclaims that follow thereafter. The present compounds can also be used asintermediates in subsequent examples to produce additional compounds ofthe present invention. No attempt has necessarily been made to optimizethe yields obtained in any of the reactions. One skilled in the artwould know how to increase such yields through routine variations inreaction times, temperatures, solvents, reagents and chemical synthesisother parameters.

The present invention is further illustrated in Examples 1, which showspreparative methods for synthesizing CDNs, and in Example 2, which showsmethods for the biological evaluation of these CDNs. It will beunderstood by one of ordinary skill in the art that these examples arein no way limiting and that variations of detail can be made withoutdeparting from the spirit and scope of the present invention.

The terms used in describing the invention are commonly used and knownto those skilled in the art. As used herein, the following abbreviationshave the indicated meanings:

° C. for degrees Celsius; A for adenosine; ACN for acetonitrile; aq. foraqueous; CDCl₃ for deuterated chloroform; C₁₈ for octadecyl carbon chainbonded silica; d for doublet; dA for deoxyadenosine; dd for doublet ofdoublets; dI for deoxyinosine; D₂O for deuterium oxide; DCA fordichloroacetic acid; DCM for dichloromethane; DMSO-d₆ for deuterateddimethylsulfoxide; DMTrCI for 4;4′-dimethoxytrityl chloride; equiv. forequivalent; ES for electrospray ionization; Et₂O for diethyl ether;EtOAc ethyl acetate; EtOH for ethanol; Et₃N.3HF for triethylaminetrihydrofluoride; g for grams; ¹H for proton; h for hours; Hz for Hertz;HPLC for high-performance liquid chromatography; I for inosine; IFN forinterferon; IFN-α for interferon alpha; IFN-β for interferon beta; iPrOHfor isopropanol; IRF3 for interferon regulatory factor 3; ISG (or ISG54)for interferon-stimulated gene; ISRE for interferon-stimulated responseelement; i.v. for intravenous; LC for liquid chromatography; m formultiplet; M for molar; m/z for mass-to-charge ratio; MeOH for methanol;mg for milligrams; MgSO₄ for magnesium sulfate; MHz for megahertz; minfor Minutes; mL (or ml) for milliliters; mmol for millimoles; mol/L formole/liter; MS for mass spectrometry; NaHCO₃ for sodium bicarbonate;NaHSO₃ for sodium thiosulfate; NH₄OH for ammonium hydroxide; NF-κB fornuclear factor kappa-light-chain-enhancer of activated B cells; NMR fornuclear magnetic resonance; PADS for phenylacetyl disulfide; ppm forparts per million; PPTS for pyridinium p-toluenesulfonate; rt for roomtemperature; SEAP for secreted embryonic alkaline phosphatase; s forsinglet; sl for large singlet; t for triplet; SKO for homozygous STINGknockout; STING for stimulator of interferon genes; TBAF fortetra-n-butylammonium fluoride; THF for tetrahydrofuran; TBDMSCl fortert-butyldimethylsilyl chloride; TEAA for triethylammonium acetate; TFAfor trifluoroacetic acid; TIPSCl2 for1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane; WT for wild type; μg formicrogram; μL (or μl) for microliter; μm for micrometer; δ for chemicalshift.

Anhydrous solvents and reagents suitable for nucleoside and nucleotidesynthesis were purchased and were handled under dry argon or nitrogenusing anhydrous technique. Amidite coupling reactions and cyclizationswere performed in anhydrous acetonitrile or pyridine under dry argon ornitrogen. The starting materials for all reactions in dry pyridine weredried by concentration (three times) from pyridine. Preparativesilica-gel flash chromatography was performed using Fluka 60 Åhigh-purity grade or Merck Grade 9385 silica using gradients of methanolin dichloromethane. Analytical LC/ES MS was performed on an Agilent 1290Infinity UHPLC system coupled to a diode array detector (DAD) Agilent1260 Infinity and an Agilent 6130 Quadrupole mass spectrometer equippedwith an electrospray ionization source (ESI) and controlled byChemstation software. The LC system was equipped with an Aquity CSH C1850×2.1 mm 1.7 μm column using gradients of 10 mM ammonioum formate andacetonitrile at 300 μl/min flow. The UV detection wavelength was 254 nm.The mass spectrometer was operated in positive and negative ESI modesPreparative HPLC was performed on a Waters preparative 150Q HPLC systemmonitoring at 254 nm on a SunFire Prep C18 5 μm OBD 30×150 mm columnusing gradients of 10 mM ammonium formate and acetonitrile at a flowrate 60 mL/min. The ¹H NMR spectra were acquired on either a Bruker 300MHz (Fourrier 300) at room temperature and reported in ppm downfield.Molecular sieves (MS) 3 Å were employed after drying the commerciallysupplied product at 250° C. for 12 h under vacuum. The commercialnucleoside phosphoramidites were supplied from Chemgenes.

The following examples serve to illustrate the present invention. Theseexamples are in no way intended to limit the scope of the invention.

Example 1: Synthesis of the Compounds of the Invention Example 1.A:General Protocol for Preparation of Phosphotriester

The appropriately protected phosphoramidite or commercially availablephosphoramidite was coevaporated three times with dry ACN, and theresulting solid was dissolved in a solution of Activator42® (0.1 mol/L,2 equiv.) in the presence of molecular sieves 3 Å. To the solution wasadded allyl alcohol (2 equiv.) and the resulting mixture was stirred for30 min.

For Phosphotriester Linkage:

Tert-butyl hydroperoxide in decane (5.5 M, 2 equiv.) was added to themixture, which was stirred for 40 min. The solution was filtered and themolecular sieves were washed with DCM. The filtrate was concentrated invacuo.

For Phosphorothioate Triester Linkage:

The mixture was concentrated in vacuo and the residue was dissolved in asolution of PADS (2.5 equ) 0.2 M in dry pyridine. The mixture wasstirred at rt for 45 min. The solution was filtered and the molecularsieves were washed with DCM. The filtrate was concentrated in vacuo andcoevaporated three times with ACN.

The residue was treated with a solution of DCA/DCM (3%) in the presenceof water (10 equiv.) for 15 min. The reaction was quenched with additionof MeOH and pyridine. The solvents were removed in vacuo and the residuewas purified by silica-gel column chromatography, using DCM/MeOH aseluent. The structure of the compound was confirmed by LC-ES/MS analysiswith ions at [M−H]⁻ and/or [M+H]⁺.

Example 1.B: General Protocol for Preparation of H-Phosphonate

The appropriately protected phosphoramidite or commercialphosphoramidite was dissolved in a solution of ACN. To the solution wereadded water (2 equiv.) and pyridium TFA (1.2 equiv.), and the mixturewas stirred for 15 min. Then, the solvents were removed in vacuo. Theresidue was treated with a solution of DCA/DCM (3%) in the presence ofwater (10 equiv.) for 15 min. The reaction was quenched with addition ofMeOH and pyridine. The solvents were removed in vacuo and the residuewas purified by silica-gel column chromatography, using DCM/MeOH aseluent. The structure of the compound was confirmed by LC-ES/MS analysiswith ions at [M−H]⁻ and/or [M+H]⁺.

Example 1.C: Protocol for the Dinucleotide Synthesis

To a solution of compound of Example 1.A or appropriately protectedcompound in a solution of Activator42® (0.1 mol/L, 2 equiv.) in thepresence of molecular sieves 3 Å was added in one portion anappropriately protected phosphoramidite or commercial phosphoramidite.The mixture was stirred for 30 min.

For Phosphotriester Linkage:

Tert-butyl hydroperoxide in decane (5.5 M, 2 equiv.) was added to themixture, which was stirred for 40 min. The solution was filtered and themolecular sieves were washed with DCM. The filtrate was concentrated invacuo.

For Phosphorothioate Triester Linkage:

The mixture was concentrated in vacuo and the residue was dissolved in asolution of PADS (2.5 equ) 0.2 M in dry pyridine. The mixture wasstirred at rt for 45 min. The solution was filtered and the molecularsieves were washed with DCM. The filtrate was concentrated in vacuo andcoevaporated three times with ACN.

The residue was treated with a solution of DCA/DCM (3%) in the presenceof water (10 equiv.) for 15 min. The reaction was quenched by additionof MeOH and pyridine. The solvents were removed in vacuo and the residuewas purified by silica-gel column chromatography, using DCM/MeOH aseluent. The structure of the compound was confirmed by LC-ES/MS analysiswith ions at [M−H]⁻ and/or [M+H]⁺.

Example 1.D: Alternative Protocol for the Dinucleotide Synthesis

To a solution of compound of Example 1.B in a solution of Activator42®(0.1 mol/L, 2 equiv.) in the presence of molecular sieves 3 Å was addedin one portion an appropriately protected phosphoramidite or commercialphosphoramidite. The mixture was stirred for 30 min.

For Phosphotriester Linkage:

Tert-butyl hydroperoxide in decane (5.5 M, 2 equiv.) was added to themixture, which was stirred for 40 min. The solution was filtered and themolecular sieves were washed with DCM. The filtrate was concentrated invacuo.

For Phosphorothioate Triester Linkage:

The mixture was concentrated in vacuo and the residue was dissolved in asolution of PADS (2.5 equ) 0.2 M in dry pyridine. The mixture wasstirred at rt for 45 min. The solution was filtered and the molecularsieves were washed with DCM. The filtrate was concentrated in vacuo andcoevaporated three times with ACN.

The residue was treated with a solution of DCA/DCM (3%) in the presenceof water (10 equiv.) for 15 min. The reaction was quenched with additionof MeOH and pyridine. The solvents were removed in vacuo and the residuewas purified by silica-gel column chromatography, using DCM/MeOH aseluent. The structure of the compound was confirmed by LC-ES/MS analysiswith ions at [M−H]⁻ and/or [M+H]⁺.

Example 1.E: Protocol to Remove Allyl Group

To a solution of dinucleotide from Example 1.C in acetone was addedsodium iodide (10 equiv.), and the resulting suspension was stirredunder reflux for 2 h. The resulting colorless precipitate was collectedby filtration and washed with chilled acetone. This precipitate ishighly hygroscopic and thus, was immediately used in the next procedure.The structure of the compound was confirmed by LC-ES/MS analysis withions at [M−H]⁻ and/or [M+H]⁺.

Example 1.F: Alternative Protocol to Remove Allyl Group

To a solution of dinucleotide from Example 1.C in dry THF was addedN-methyl aniline (3 equiv.) and tetrakis(triphenylphosphine)palladium(0)(0.2 equiv.). The resulting suspension was stirred at rt for 15 min.Then, the solvent was removed in vacuo and the residue was trituratedwith diethyl ether. The resulting colorless precipitate was collected byfiltration and washed with chilled diethyl ether. This precipitate waspurified by silica-gel column chromatography, using DCM/MeOH as eluent.The structure of the compound was confirmed by LC-ES/MS analysis withions at [M−H]⁻ and/or [M+H]⁺.

Example 1.G: Protocol for Cyclization of the Dinucleotide

The solid obtained in Example 1.E or 1.F was coevaporated three timeswith dry pyridine and then, dry ACN. The residue was suspended in THF,and to the resulting heterogeneous mixture were successively addedN-methylimidazole (10 equiv.) and 2,4,6-triisopropylbenzenesulfonylchloride (10 equiv.). The resulting mixture was stirred at 25° C. for 3h to 36 h. Then, the solvent was removed in vacuo and the residue wastriturated with EtOAc. The resulting colorless precipitate was collectedby filtration and washed with chilled EtOAc. This precipitate was usedin the next step without any further purification. The structure of thecompound was confirmed by LC-ES/MS analysis with ions at [M−H]⁻ and/or[M+H]⁺.

Example 1.H: Alternative Protocol for Cyclization of the Dinucleotide

The solid obtained from Example 1.E or 1.F was coevaporated three timeswith dry pyridine. The residue was suspended in dry pyridine, and to theresulting solution was added1-(Mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole (MSNT) (5 equiv.). Theresulting mixture was stirred at 25° C. for 3 h to 18 h. Then, thesolvent was removed in vacuo, and the resulting product used in the nextstep without any further purification. The structure of the compound wasconfirmed by LC-ES/MS analysis with ions at [M−H]⁻ and/or [M+H]⁺.

Example 1.1: Alternative Protocol for Cyclization of the Dinucleotide

The solid obtained in Example 1.D or appropriately protected compoundwas coevaporated three times with dry pyridine. The residue wassuspended in dry pyridine, and to the resulting solution was added5,5-dimethyl-2-oxo-2-chloro-1,3,2-dioxaphosphinane (DMOCP) (3 equiv.).The resulting mixture was stirred at 25° C. for 3 h to 18 h.

For Phosphodiester Linkage:

Iodine (1.3 equiv.) and water (30 equiv.) were added to the mixture.After 10 min aq. NaHSO₃ (0.15%) was added until complete decolorationwas observed, and then aq. NaHCO₃ was added. The aqueous layer wasextracted three times with a 1:1 (v/v) mixture of EtOAc/Et₂O. Theorganic layers were pooled, dried over MgSO₄, filtered, and thenconcentrated in vacuo.

For Phosphorothioate Triester Linkage:

Elemental sulfur (5 equiv.) was added. The mixture was stirred at rt for45 min. Then the mixture was concentrated in vacuo and coevaporatedthree times with toluene, precipitated in ACN to removed excess ofsulfur and concentrated in dryness.

The residue was used in the next step without any further purification.The structure of the compound was confirmed by LC-ES/MS analysis withions at [M−H]⁻ and/or [M+H]⁺.

Example 1.J: Protocol for the Deprotection and Purification of CyclicDinucleotides

The protected cyclic dinucleotide of Example 1.G, 1.H or 1.1 was treatedwith a solution of methylamine in EtOH (33%), and the resulting mixturewas stirred at 50° C. for 4 h. The reaction mixture was concentrated,and the resulting residue was dried in vacuo. The dried material wasmixed with Et₃N.3HF (25 equiv.) and stirred at 25° C. for 6 h. To thismixture was added a 1 M ammonium formate buffer solution, and themixture was vigorously stirred at 30° C. to 40° C. for 10 min. Theresulting precipitate was filtered, and the filtrate was subjected topreparative HPLC using a C₁₈ Sunfire column (19×150 mm, 5 μm) andammonium formate/ACN as eluent. The fractions containing the desiredcompound were pooled and lyophilized. The structure of the compound wasconfirmed by LC-ES/MS analysis with ions at [M−H]⁻ and/or [M+H]⁺.

Example 1.K: Protocol for the Deprotection and Purification of CyclicDinucleotides

The protected cyclic dinucleotide of Example 1.G, 1.H or 1.1 was treatedwith a solution of methylamine in EtOH (33%), and the resulting mixturewas stirred at 50° C. for 4 h. The reaction mixture was concentrated,and the resulting residue was subjected to preparative HPLC using a C₁₈Sunfire column (19×150 mm, 5 μm) and ammonium formate/ACN as eluent. Thefractions containing the desired compound were pooled and lyophilized.The structure of the compound was confirmed by LC-ES/MS analysis withions at [M−H]⁻ and/or [M+H]⁺.

Intermediate 1

Intermediate 1 was prepared from the commercially availablephosphoramidite of Adenosine using a similar procedure to that describedin Example 1.B to provide 6.20 g (93% yield) of intermediate 1. LC-MS:Rt=4.41 min, m/z=550 [M+H]⁺, m/z=548 [M−H]⁻

Intermediate 2

Intermediate 2 was prepared from intermediate 1 and the commerciallyavailable phosphoramidite of Inosine using a similar procedure to thatdescribed in Example 1.D to provide 8.04 g (68% yield) of intermediate2. LC-MS: Rt=5.22 min, m/z=1048 [M+H]⁺, m/z=1046 [M−H]⁻.

Intermediate 3

Intermediate 3 was prepared from intermediate 2 using a similarprocedure to that described in Example 1.1 to provide 8.04 g (68% yield)of intermediate 3. LC-MS: Rt=5.41 min, m/z=1046 [M+H]⁺, m/z=1044 [M−H]⁻.

Intermediate 4

To a solution of Inosine (10.0 g, 37.2 mmol) in dry pyridine (200 mL)was added TIPSCl₂ (14.1 g, 44.7 mmol). The solution was stirred for 18 hat rt. Then, the reaction was quenched by addition of MeOH (50 mL) andthe solvents were removed in vacuo. The residue was dissolved in EtOAcand washed with saturated aq. NaHCO₃, water and brine. The organic layerwas dried over MgSO₄, filtered, and then concentrated in vacuo. Thecrude compound was purified by silica-gel column chromatography, usingDCM/MeOH as eluent, to give 13.5 g (70% yield) intermediate 4. LC-MS:Rt=5.32 min, m/z=511 [M+H]⁺, m/z=509 [M−H]⁻. ¹H NMR (CDCl₃-d₁, 300 MHz)δ (ppm) 13.04 (s, 1H), 8.10 (s, 1H), 8.03 (s, 1H), 5.99 (s, 1H), 4.93(t, 1H), 4.50 (d, 1H), 4.11 (m, 3H), 1.83 (m, 4H), 1.09 (m, 32H).

Intermediate 5

To a solution of intermediate 4 (5 g, 9.79 mmol) in dry DCM (75 mL) wereadded 3,4-dihydro-2H-pyran (24.7 g, 293.7 mmol) and PPTS (7.38 g, 29.37mmol). The solution was stirred for 18 h at rt. Then, the reaction wasquenched by a saturated solution of NaHCO₃. The different layers wereseparated and the organic layer was washed with water and brine, driedover MgSO₄, filtered, and then concentrated in vacuo. The crude compoundwas purified by silica-gel column chromatography, using DCM/MeOH aseluent, to give 4.71 g (80% yield) of intermediate 5. LC-MS: Rt=6.72min, m/z=595 [M+H]⁺, m/z=593 [M−H]⁻. ¹H NMR (CDCl₃-d₁, 300 MHz) δ (ppm)12.89 (d, 1H), 8.12 (s, 1H), 8.09 (s, 1H), 8.04 (d, 1H), 6.05 (d, 1H),5.07 (t, 1H), 4.76 (m, 1H), 4.53 (m, 2H), 4.40-4.05 (m, 5H), 1.85-1.55(m, 10H), 1.04 (m, 32H).

Intermediate 6

To a solution of intermediate 5 (4.71 g, 7.92 mmol) in THF (100 mL) wasadded TBAF on silica gel (10.56 g, 15.84 mmol). The solution was stirredfor 3 h at rt. Then, the reaction was filtered, the silica gel waswashed with THF, and the filtrate was concentrated in vacuo. The crudecompound was purified by silica-gel column chromatography, usingDCM/MeOH as eluent, to give 2.7 g (96% yield) of intermediate 6. LC-MS:Rt=4.32 min, m/z=353 [M+H]⁺, m/z=351 [M−H]⁻.

Intermediate 7

To a solution of intermediate 6 (2.70 g, 7.66 mmol) in dry pyridine (40mL) was added dropwise a solution of DMTrCI (2.17 g, 6.42 mmol) in DCM(5 mL). The solution was at rt for 2 h. Then, the reaction was quenchedby addition of 5% aq. NaHCO₃ (110 mL), the aqueous layer was extractedthree times with DCM. The organic layers were pooled and dried overMgSO₄, filtered, and then concentrated in vacuo. The residue waspurified by silica-gel column chromatography, using 1% pyridine inDCM/MeOH as eluent, to give 4.78 g (95% yield) of intermediate 7. LC-MS:Rt=6.80 min, m/z=655 [M+H]⁺, m/z=653 [M−H]⁻. ¹H NMR (CDCl₃-d₁, 300 MHz)δ (ppm) 12.80 (s, 1H), 8.02 (s, 1H), 8.00 (s, 1H), 7.71 (m, 2H), 7.73(m, 2H), 7.24 (m, 9H), 6.20 (d, 1H), 5.07 (t, 1H), 4.83 (m, 1H), 4.35(m, 2H), 3.78 (s, 6H), 3.45-3.30 (m, 4H), 1.77-1.56 (m, 6H).

Intermediate 8

To a solution of intermediate 7 (4.78 g, 7.30 mmol) in dry pyridine (40mL) were added imidazole (1.29 g, 18.98 mmol) and TBDMSCl (1.43 g, 9.49mmol), the reaction was stirred for 18 h at rt. Then, the reaction wasdiluted with DCM (100 mL), the solution was washed with saturated aq.NaHCO₃, water and brine. The organic layer was dried over MgSO₄,filtered, and then concentrated in vacuo. The residue was purified bysilica-gel column chromatography, using 1% pyridine in DCM/MeOH aseluent, to give ((5.12 g (91% yield) of intermediate 8. LC-MS: Rt=8.15min, m/z=769 [M+H]⁺, m/z=767 [M−H]⁻. 1H NMR (CDCl₃-d₁, 300 MHz) δ (ppm)12.70 (s, 1H), 8.02 (s, 1H), 7.97 (s, 1H), 7.40 (m, 2H), 7.26 (m, 11H),6.09 (d, 1H), 4.80 (m, 1H), 4.36 (m, 1H), 4.16 (m, 2H), 3.72 (s, 6H),3.45-3.26 (m, 4H), 1.61-1.37 (m, 8H), 0.81 (s, 9H), 0.09 (dd, 6H).

Intermediate 9

The intermediate 8 (4.93 g, 6.41 mmol) was dissolved in a solution ofZnBr₂ (0.5 M) in DCM/iPrOH (1/1) (40 mL, 19.3 mmol). The solution wasstirred for 40 min at rt. The reaction was neutralized with 1N NaHCO₃solution. The different layers were separated and the organic layer waswashed with water and brine, dried over MgSO₄, filtered, and thenconcentrated in vacuo. The residue was purified by silica-gel columnchromatography, using DCM/MeOH as eluent to give 2.68 g (90% yield) ofintermediate 9. LC-MS: Rt=6.22 min, m/z=467 [M+H]⁺, m/z=465 [M−H]⁻.

Intermediate 10

Intermediate 10 was prepared from intermediate 9 and the commerciallyavailable phosphoramidite of Adenosine using a similar procedure to thatdescribed in Example 1.C to give 1.9 g (64% yield) of intermediate 10.Rt=6.99 min, m/z=1068 [M+H]⁺, m/z=1066 [M−H]⁻.

Intermediate 11

To a solution of intermediate 10 (1.9 g, 1.78 mmol) in dry pyridine (20mL) was added diphenylphosphite (1.25 g, 5.34 mmol). The reaction wasstirred for 2 h at rt. To the reaction was added a solution of TEAA 0.1M (53 mL, 5.34 mmol). The reaction was stirred for 45 min at rt. Then,the solvents were removed in vacuo and the residue was dissolved in DCM(100 mL). The organic layer was washed with aq. NaHCO₃ (5%) solution,water and brine, dried over MgSO₄, filtered, and then concentrated invacuo to give 2.0 g (99% yield) of crude intermediate 11. Thisintermediate was used in the next step without any further purification.LC-MS: Rt=6.06 min, m/z=1132 [M+H]⁺, m/z=1130 [M−H]⁻.

Intermediate 12

The intermediate 11 (2.0 g, 2.12 mmol) was treated with a solution ofDCA 10% in DCM (50 mL). The reaction was stirred for 2 h at rt. Then,the reaction was neutralized by addition of pyridine (17 mL). Thesolvents were removed in vacuo to give 2.2 g (100% yield) of crudeintermediate 12. This intermediate was used in the next step without anyfurther purification. LC-MS: Rt=7.38 min, m/z=1048 [M+H]⁺, m/z=1046[M−H]⁻.

Intermediate 13

Intermediate 13 was prepared from intermediate 12 using a similarprocedure to that described in Example 1.1 to provide 1.73 g (83% yield)of intermediate 13. LC-MS: Rt=7.20 min, m/z=1046 [M+H]⁺, m/z=1044[M−H]⁻.

Intermediate 14

Intermediate 14 was prepared from intermediate 9 and the commerciallyavailable phosphoramidite of adenosine using a similar procedure to thatdescribed in Example 1.C to provide 1.9 g (60% yield) of intermediate14. Rt=7.08 min, m/z=1068 [M+H]⁺, m/z=1066 [M−H]⁻.

Intermediate 15

To a solution of intermediate 14 (1.9 g, 1.78 mmol) in dry pyridine (20mL) was added diphenylphosphite (1.25 g, 5.34 mmol). The reaction wasstirred at rt for 2 h. To the reaction was added a solution of TEAA 0.1M (53 mL, 5.34 mmol). The reaction was stirred for 45 min at rt. Then,the solvents were removed in vacuo and the residue was dissolved in DCM(100 mL). The organic layer was washed with aq. NaHCO₃ (5%) solution,water and brine, dried over MgSO₄, filtered, and then concentrated invacuo to give 2.0 g (99% yield) of crude intermediate 15. Thisintermediate was used in the next step without any further purification.LC-MS: Rt=6.11 min, m/z=1132 [M+H]⁺, m/z=1130 [M−H]⁻.

Intermediate 16

The intermediate 15 (2.0 g, 1.76 mmol) was treated with a solution ofDCA 10% in DCM (50 mL, 85.0 mmol). The reaction was stirred at rt for 2h. Then, the reaction was neutralized by addition of pyridine (17 mL,177.0 mmol). The solvents were removed in vacuo to give 1.8 g (100%yield) of crude intermediate 16. This intermediate was used in the nextstep without any further purification. LC-MS: Rt=5.39 min, m/z=1048[M+H]⁺, m/z=1046 [M−H]⁻.

Intermediate 17

Intermediate 17 was prepared from intermediate 16 using a similarprocedure to that described in Example 1.1 to provide 1.5 g (75% yield)of intermediate 17. LC-MS: Rt=5.41 min, m/z=1046 [M+H]⁺, m/z=1044[M−H]⁻.

Intermediate 18

Intermediate 18 was prepared from the commercially availablephosphoramidite of Adenosine using a similar procedure to that describedin Example 1.B to provide 1.1 g (94% yield) of intermediate 18. LC-MS:Rt=4.47 min, m/z=550 [M+H]⁺, m/z=548 [M−H]⁻.

Intermediate 19

Intermediate 19 was prepared from intermediate 18 and commerciallyavailable phosphoramidite of Inosine using a similar procedure to thatdescribed in Example 1.D to provide 410 mg (44% yield) of intermediate19. Rt=4.70 min, m/z=1048 [M+H]⁺, m/z=1046 [M−H]⁻.

Intermediate 20

Intermediate 20 was prepared from intermediate 19 using a similarprocedure to that described in Example 1.1 to provide 364 mg (89% yield)of intermediate 20. LC-MS: Rt=5.66 min, m/z=1046 [M+H]⁺, m/z=1044[M−H]⁻.

Intermediate 21

Intermediate 21 was prepared from intermediate 1 and the commerciallyavailable phosphoramidite of Inosine using a similar procedure to thatdescribed in Example 1.D to provide 1.05 g (65% yield) of intermediate21. LC-MS: Rt=5.42 and 5.52 min, m/z=1064 [M+H]⁺, m/z=1062 [M−H]⁻.

Intermediate 22

Intermediate 22 was prepared from intermediate 21 using a similarprocedure to that described in Example 1.1 to provide 579 mg (55% yield)of intermediate 22. LC-MS: Rt=5.61 and 5.71 min, m/z=1078 [M+H]⁺,m/z=1076 [M−H]⁻.

Intermediate 23

Intermediate 23 was prepared from the commercially availablephosphoramidite of 2′-deoxyadenosine using a similar procedure to thatdescribed in Example 1.A to provide 1.84 g (95% yield) of intermediate23. LC-MS: Rt=4.31 min, m/z=529 [M+H]⁺, m/z=527 [M−H]⁻.

Intermediate 24

Intermediate 24 was prepared from intermediate 23 and commerciallyavailable phosphoramidite of 2′-deoxyinosine using a similar procedureto that described in Example 1.C to provide 500 mg (32% yield) ofintermediate 24. Rt=5.28 min, m/z=896 [M+H]⁺, m/z=894 [M−H]⁻.

Intermediate 25

Intermediate 25 was prepared from intermediate 24 using a similarprocedure to that described in Example 1.E to provide 390 mg (83% yield)of intermediate 25. LC-MS: Rt=3.35 min, m/z=856 [M+H]⁺, m/z=854 [M−H]⁻.

Intermediate 26

Intermediate 26 was prepared from intermediate 25 using a similarprocedure to that described in Example 1.G to provide 0.38 g (99% yield)of intermediate 26. LC-MS: Rt=3.91 min, m/z=838 [M+H]⁺, m/z=836 [M−H]⁻.

Intermediate 27

Intermediate 27 was prepared from intermediate 23 and commerciallyavailable phosphoramidite of 2′-deoxy-2′-fluorolnosine using a similarprocedure to that described in Example 1.C to provide 3.16 g (62% yield)of intermediate 27. Rt=4.27 min, m/z=914 [M+H]⁺, m/z=912 [M−H]⁻.

Intermediate 28

Intermediate 28 was prepared from intermediate 27 using a similarprocedure to that described in Example 1.E to provide 1.10 g (69% yield)of intermediate 28. LC-MS: Rt=3.52 min, m/z=874 [M+H]⁺, m/z=872 [M−H]⁻.

Intermediate 29

Intermediate 29 was prepared from intermediate 28 using a similarprocedure to that described in Example 1.H to provide 1.01 g (99% yield)of intermediate 29. LC-MS: Rt=4.23 and 4.07 min, m/z=856 [M+H]⁺, m/z=854[M−H]⁻.

Intermediate 30

Intermediate 30 was prepared from the commercially availablephosphoramidite of 2′-deoxy-2′-fluoroAdenosine using a similar procedureto that described in Example 1.A to provide 2.68 g (85% yield) ofintermediate 30. LC-MS: Rt=4.50 min, m/z=547 [M+H]⁺, m/z=545 [M−H]⁻.

Intermediate 31

Intermediate 31 was prepared from intermediate 30 and commercialphosphoramidite of 2′-deoxy-2′-fluorolnosine using a similar procedureto that described in Example 1.C to provide 2.53 g (55% yield) ofintermediate 31. Rt=4.39 min, m/z=932 [M+H]⁺, m/z=930 [M−H]⁻.

Intermediate 32

Intermediate 32 was prepared from intermediate 31 using a similarprocedure to that described in Example 1.E to provide 4.4 g (92% yield)of intermediate 32. LC-MS: Rt=3.58 and 3.59 min, m/z=892 [M+H]⁺, m/z=891[M−H]⁻.

Intermediate 33

Intermediate 33 was prepared from intermediate 32 using a similarprocedure to that described in Example 1.G to provide 1.40 g (99% yield)of intermediate 33. LC-MS: Rt=4.21 and 4.42 min, m/z=874 [M+H]⁺, m/z=872[M−H]⁻.

Intermediate 34

Intermediate 34 was prepared from the commercially availablephosphoramidite of 2′-deoxy-2′-fluoroAdenosine using a similar procedureto that described in Example 1.A to provide 647 mg (55% yield) ofintermediate 34. LC-MS: Rt=5.06 min, m/z=563 [M+H]⁺, m/z=561 [M−H]⁻.

Intermediate 35

Intermediate 35 was prepared from intermediate 34 and commerciallyavailable phosphoramidite of 2′-deoxy-2′-fluorolnosine using a similarprocedure to that described in Example 1.C to provide 447 mg (35% yield)of intermediate 35. Rt=6.20 min, m/z=964 [M+H]⁺, m/z=962 [M−H]⁻.

Intermediate 36

Intermediate 36 was prepared from intermediate 35 using a similarprocedure to that described in Example 1.E to provide 232 mg (43% yield)of intermediate 36. LC-MS: Rt=4.25 and 4.55 min, m/z=924 [M+H]⁺, m/z=922[M−H]⁻.

Intermediate 37

Intermediate 37 was prepared from intermediate 36 using a similarprocedure to that described in Example 1.G to provide 35 mg (36% yield)of intermediate 37. LC-MS: Rt=5.00 and 5.32 min, m/z=906 [M+H]⁺, m/z=904[M−H]⁻.

Intermediate 38

Intermediate 38 was prepared from the commercially available2′-phosphoramidite of Adenosine using a similar procedure to thatdescribed in Example 1.B to provide 1.81 g (95% yield) of intermediate38. LC-MS: Rt=4.53 min, m/z=550 [M+H]⁺, m/z=548 [M−H]⁻.

Intermediate 39

Intermediate 39 was prepared from intermediate 38 and commerciallyavailable phosphoramidite of 2′-deoxy-2′-fluorolnosine using a similarprocedure to that described in Example 1.D to provide 340 mg (55% yield)of intermediate 39. Rt=4.28 min, m/z=935 [M+H]⁺, m/z=933 [M−H]⁻.

Intermediate 40

Intermediate 40 was prepared from intermediate 39 using a similarprocedure to that described in Example 1.1 to provide 340 mg (95% yield)of intermediate 40. LC-MS: Rt=4.44 min, m/z=933 [M+H]⁺, m/z=931 [M−H]⁻.

Intermediate 41

Intermediate 41 was prepared from the commercially availablephosphoramidite of 2′-deoxy-2′-fluorolnosine using a similar procedureto that described in Example 1.A to provide 600 mg (98% yield) ofintermediate 41. LC-MS: Rt=3.78 min, m/z=444 [M+H]⁺, m/z=442 [M−H]⁻.

Intermediate 42

Intermediate 42 was prepared from intermediate 41 and commerciallyavailable phosphoramidite of 2′-deoxy-2′-fluorolnosine using a similarprocedure to that described in Example 1.C to provide 610 mg (55% yield)of intermediate 42. Rt=5.50 min, m/z=829 [M+H]⁺, m/z=827 [M−H]⁻.

Intermediate 43

Intermediate 43 was prepared from intermediate 42 using a similarprocedure to that described in Example 1.F to provide 580 mg (90% yield)of intermediate 43. LC-MS: Rt=3.40 min, m/z=789 [M+H]⁺, m/z=787 [M−H]⁻.

Intermediate 44

Intermediate 44 was prepared from intermediate 43 using a similarprocedure to that described in Example 1.H to provide 500 mg (99% yield)of intermediate 44. LC-MS: Rt=3.86 min, m/z=771 [M+H]⁺, m/z=769 [M−H]⁻.

Intermediate 45

Intermediate 45 was prepared from the commercially availablephosphoramidite of 2′-deoxy-2′-fluoroGuanosine using a similar procedureto that described in Example 1.A to provide 3.75 g (60% yield) ofintermediate 45. LC-MS: Rt=4.25 min, m/z=529 [M+H]⁺, m/z=527 [M−H]⁻.

Intermediate 46

Intermediate 46 was prepared from intermediate 45 and commerciallyavailable phosphoramidite of 2′-deoxy-2′-fluoroGuanosine using a similarprocedure to that described in Example 1.C to provide 1.07 g (67% yield)of intermediate 46. Rt=4.80 min, m/z=999 [M+H]⁺, m/z=997 [M−H]⁻.

Intermediate 47

Intermediate 47 was prepared from intermediate 46 using a similarprocedure to that described in Example 1.F to provide 610 mg (99% yield)of intermediate 47. LC-MS: Rt=4.05 min, m/z=959 [M+H]⁺, m/z=957 [M−H]⁻.

Intermediate 48

Intermediate 48 was prepared from intermediate 47 using a similarprocedure to that described in Example 1.H to provide 580 mg (96% yield)of intermediate 48. LC-MS: Rt=4.77 min, m/z=941 [M+H]⁺, m/z=939 [M−H]⁻.

Intermediate 49

Intermediate 49 was prepared from intermediate 45 and commerciallyavailable phosphoramidite of 2′-deoxy-2′-fluoroAdenosine using a similarprocedure to that described in Example 1.C to provide 4.96 g (68% yield)of intermediate 49. Rt=4.98 min, m/z=1017 [M+H]⁺, m/z=1015 [M−H]⁻.

Intermediate 50

Intermediate 50 was prepared from intermediate 47 using a similarprocedure to that described in Example 1.E to provide 4.4 g (92% yield)of intermediate 50. LC-MS: Rt=4.36 min, m/z=977 [M+H]⁺, m/z=975 [M−H]⁻.

Intermediate 51

Intermediate 51 was prepared from intermediate 50 using a similarprocedure to that described in Example 1.G to provide 4.30 g (99% yield)of intermediate 51. LC-MS: Rt=5.72 min, m/z=838 [M+H]⁺, m/z=836 [M−H]⁻.

Intermediate 52

Intermediate 52 was prepared from the commercially availablephosphoramidite of 2′-deoxy-2′-fluoroGuanosine using a similar procedureto that described in Example 1.A to provide 632 mg (50% yield) ofintermediate 52. LC-MS: Rt=5.86 min, m/z=545 [M+H]⁺, m/z=543 [M−H]⁻.

Intermediate 53

Intermediate 53 was prepared from intermediate 52 and commerciallyavailable phosphoramidite of 2′-deoxy-2′-fluoroAdenosine using a similarprocedure to that described in Example 1.C to provide 310 mg (25% yield)of intermediate 53. Rt=6.60 min, m/z=1049 [M+H]⁺, m/z=1047 [M−H]⁻.

Intermediate 54

Intermediate 54 was prepared from intermediate 53 using a similarprocedure to that described in Example 1.E to provide 100 mg (33% yield)of intermediate 54. LC-MS:Rt=4.58 and 4.70 min, m/z=1009 [M+H]⁺,m/z=1007 [M−H]⁻.

Intermediate 55

Intermediate 55 was prepared from intermediate 54 using a similarprocedure to that described in Example 1.G to provide 75 mg (76% yield)of intermediate 55. LC-MS: Rt=5.32 and 5.54 min, m/z=991 [M+H]⁺, m/z=989[M−H]⁻.

Intermediate 56

A solution of chloromethyl pivalate (1.0 g, 6.64 mmol) in dry ACN (15mL) was treated with sodium iodide (1.9 g, 13.28 mmol). The mixture wasstirred at RT overnight in the dark. Then the solvent was removed invacuo and the residue was dissolved in DCM. The solution was washed withwater, 5% NaHSO₃ solution and brine. The organic layer was dried overMgSO4, filtered and concentrated in vacuo to provide 1.22 g of crudeintermediate 53 which was used for the next step without any furtherpurification. ¹H NMR (CDCl₃-d₁, 300 MHz) δ (ppm) 5.86 (s, 2H), 1.12 (s,9H).

Example 1.1

Example 1.1 was prepared from intermediate 3 using a similar procedureto that described in Example 1.J to provide 2.4 g (60% yield) of example1.1. LC-MS: Rt=2.72 min, m/z=660 [M+H]⁺, m/z=658 [M−H]⁻. ¹H NMR (D₂O,300 MHz) δ (ppm) 8.34 (s, 1H), 8.27 (s, 1H), 8.10 (s, 1H), 7.86 (s, 1H),5.94 (s, 2H), 5.06-4.80 (m, 4H), 4.42 (m, 4H), 4.03 (m, 2H).

Example 1.2

Example 1.2 was prepared from intermediate 13 using a similar procedureto that described in Example 1.J to provide 22.5 mg (21% yield) ofexample 1.2. LC-MS: Rt=2.46 min, m/z=660 [M+H]⁺, m/z=658 [M−H]⁻. ¹H NMR(D₂O, 300 MHz) δ (ppm) 8.27 (s, 1H), 8.15 (s, 1H), 8.04 (s, 1H), 7.88(s, 1H), 5.99 (s, 2H), 4.95 (m, 2H), 4.86 (m, 1H), 4.42 (m, 4H), 4.05(m, 2H).

Example 1.3

Example 1.3 was prepared from intermediate 17 using a similar procedureto that described in Example 1.J to provide 17.5 mg (22% yield) ofexample 1.3. LC-MS: Rt=1.35 min, m/z=660 [M+H]⁺, m/z=658 [M−H]⁻. ¹H NMR(D₂O, 300 MHz) δ (ppm) 8.44 (s, 1H), 8.13 (s, 1H), 8.08 (s, 1H), 8.07(s, 1H), 6.20 (d, 1H), 6.07 (d, 1H), 5.22 (m, 1H), 4.91 (m, 1H), 4.79(m, 2H), 4.63 (m, 1H), 4.56 (m, 1H), 4.22 (m, 2H), 4.07 (m, 2H).

Example 1.4

Example 1.4 was prepared from intermediate 20 using a similar procedureto that described in Example 1.J to provide 50 mg (19% yield) of example1.4. LC-MS: Rt=1.53 min, m/z=660 [M+H]⁺, m/z=658 [M−H]⁻. ¹H NMR (D₂O,300 MHz) δ (ppm) 8.50 (s, 1H), 8.14 (s, 1H), 8.13 (s, 1H), 8.09 (s, 1H),6.24 (d, 1H), 6.08 (s, 1H), 5.20 (m, 1H), 4.83-4.73 (m, 2H), 4.58 (d,1H), 4.40 (m, 2H), 4.26 (m, 2H), 4.04 (m, 2H).

Example 1.5

Example 1.5 was prepared from intermediate 22 using a similar procedureto that described in Example 1.J to provide 7 mg (20% yield) of example1.5. LC-MS: Rt=3.45 min, m/z=692 [M+H]⁺, m/z=690 [M−H]⁻. ¹H NMR (D₂O,300 MHz) δ (ppm) 8.54 (s, 1H), 8.37 (s, 1H), 8.22 (s, 1H), 7.97 (s, 1H),6.65 (dd, 1H), 6.15 (dd, 1H), 4.60-4.50 (m, 4H), 4.45 (m, 4H), 4.05 (m,2H).

Example 1.6

Example 1.6 was prepared from intermediate 26 using a similar procedureto that described in Example 1.K to provide 90 mg (31% yield) of Example1.6. LC-MS: Rt=2.47 min, m/z=628 [M+H]⁺, m/z=626 [M−H]⁻. ¹H NMR(DMSO-d6, 300 MHz) δ (ppm) 8.37 (s, 1H), 8.32 (s, 1H), 8.13 (s, 1H),8.05 (s, 1H), 7.26 (sl, 2H), 6.28 (m, 2H), 4.68 (m, 2H), 4.08 (m, 2H),3.85 (m, 2H), 2.83 (m, 2H).

Example 1.7

Example 1.7 was prepared from intermediate 29 using a similar procedureto that described in Example 1.K to provide 40 mg (21% yield) of Example1.7. LC-MS: Rt=1.97 min, m/z=646 [M+H]⁺, m/z=644 [M−H]⁻. ¹H NMR (D₂O,300 MHz) δ (ppm) 8.29 (s, 1H), 8.12 (s, 1H), 7.98 (s, 1H), 7.88 (s, 1H),6.25 (m, 2H), 5.54 (m, 1H), 5.07 (m, 2H), 4.37 (m, 4H), 4.06 (m, 2H).

Example 1.8

Example 1.8 was prepared from intermediate 33 using a similar procedureto that described in Example 1.K to provide 104 mg (10% yield) ofExample 1.8. LC-MS: Rt=2.78 min, m/z=664 [M+H]⁺, m/z=662 [M−H]⁻. ¹H NMR(D₂O, 300 MHz) δ (ppm) 8.37 (s, 1H), 8.35 (s, 1H), 8.14 (s, 1H), 7.95(s, 1H), 6.23 (m, 2H), 5.45 (m, 2H), 5.39 (m, 1H), 4.95 (m, 2H), 4.50(m, 2H), 4.06 (m, 2H).

Example 1.9

Example 1.9 was prepared from intermediate 37 using a similar procedureto that described in Example 1.K to provide 10 mg (18% yield) of Example1.9. LC-MS: Rt=3.41 min, m/z=696 [M+H]⁺, m/z=694 [M−H]⁻. ¹H NMR (D₂O,300 MHz) δ (ppm) 8.55 (s, 1H), 8.38 (s, 1H), 8.20 (s, 1H), 7.99 (s, 1H),6.63 (dd, 1H), 6.15 (dd, 1H), 5.16-4.95 (m, 4H), 4.52 (m, 4H), 4.07 (m,2H).

Example 1.10

Example 1.10 was prepared from intermediate 40 using a similar procedureto that described in Example 1.J to provide 50 mg (19% yield) of Example1.10. LC-MS: Rt=2.25 min, m/z=662 [M+H]⁺, m/z=660 [M−H]⁻. ¹H NMR (D₂O,300 MHz) δ (ppm) 8.58 (s, 1H), 8.35 (s, 1H), 8.12 (s, 1H), 7.97 (s, 1H),6.65 (d, 1H), 6.16 (d, 1H), 4.50 (m, 2H), 5.15 (m, 2H), 4.41 (m, 4H),4.02 (m, 1H), 3.73 (m, 1H).

Example 1.11

Example 1.11 was prepared from intermediate 44 using a similar procedureto that described in Example 1.K to provide 157 mg (35% yield) ofExample 1.11. LC-MS: Rt=1.48 min, m/z=665 [M+H]⁺, m/z=663 [M−H]⁻. ¹H NMR(D₂O, 300 MHz) δ (ppm) 8.34 (s, 2H), 8.08 (s, 2H), 7.85 (s, 2H), 6.21(m, 2H), 5.66 (m, 1H), 5.49 (m, 1H), 5.20 (m, 2H), 4.97 (m, 4H), 4.06(m, 2H).

Example 1.12

Example 1.12 was prepared from intermediate 48 using a similar procedureto that described in Example 1.K to provide 15 mg (5% yield) of Example1.12. LC-MS: Rt=1.58 min, m/z=695 [M+H]⁺, m/z=693 [M−H]⁻. ¹H NMR (D₂O,300 MHz) δ (ppm) 8.70 (s, 2H), 6.05 (d, 2H), 5.06-4.05 (m, 8H), 4.08 (m,2H).

Example 1.13

Example 1.13 was prepared from intermediate 51 using a similar procedureto that described in Example 1.K to provide 320 mg (31% yield) ofExample 1.13. LC-MS: Rt=2.48 min, m/z=679 [M+H]⁺, m/z=677 [M−H]⁻. ¹H NMR(D₂O, 300 MHz) δ (ppm) 8.17 (s, 1H), 7.83 (s, 1H), 7.63 (s, 1H), 6.09(d, 1H), 5.94 (d, 1H), 5.62 (m, 1H), 5.50 (m, 1H), 5.15 (m, 2H), 4.42(m, 4H), 4.03 (m, 2H).

Example 1.14

Example 1.14 was prepared from intermediate 55 using a similar procedureto that described in Example 1.K to provide 15 mg (28% yield) of Example1.14. LC-MS: Rt=2.66 min, m/z=711 [M+H]⁺, m/z=709 [M−H]⁻. ¹H NMR (D₂O,300 MHz) δ (ppm) 8.58 (s, 1H), 8.35 (s, 1H), 7.97 (s, 1H), 6.65 (d, 1H),6.19 (d, 1H), 4.94 (d, 1H), 4.62-4.50 (m, 4H), 4.42 (m, 4H), 4.03 (m,2H).

Example 1.15

To a solution of Example 1.14 (7 mg, 9.85 μmol) in water (200 μL) wasadded dropwise a solution of intermediate 56 (19 mg, 79.0 μmol) inacetone (500 μL). The mixture was stirred overnight in the dark. Thenthe mixture was neutralized with a saturated solution of Na₂S₂O₃ (15 μL)and subsequently diluted with water (10 mL). The aqueous layer wasextracted three times with EtOAc (3×10 mL). The organic layers werepooled, dried over MgSO₄, filtered and concentrated in vacuo to provide4 mg (45% Yield) of Example 1.15. LC-MS: Rt=4.11 min, m/z=907 [M+H]⁺,m/z=905 [M−H]⁻. ¹H NMR (DMSO-d₆, 300 MHz) δ (ppm) 8.61 (s, 1H), 8.37 (s,1H), 8.00 (s, 1H), 6.70 (d, 1H), 6.22 (d, 1H), 4.70-4.50 (m, 4H), 4.42(m, 4H), 4.03 (m, 2H), 3.77 (s, 4H), 1.20 (s, 18H).

Example 1.16

To a solution of Example 1.9 (5 mg, 7.19 μmol) in water (200 μL) wasadded dropwise a solution of intermediate 56 (17 mg, 70 μmol) in acetone(500 μL). The mixture was stirred overnight in the dark. Then themixture was neutralized with a saturated solution of Na₂S₂O₃ (15 μL) andsubsequently diluted with water (10 mL). The aqueous layer was extractedthree times with EtOAc (3×10 mL). The organic layers were pooled, driedover MgSO₄, filtered and concentrated in vacuo to provide 5 mg (78%Yield) of Example 1.16. LC-MS: Rt=4.55 min, m/z=892 [M+H]⁺, m/z=890[M−H]⁻. ¹H NMR (DMSO-d₆, 300 MHz) δ (ppm) 8.61 (s, 1H), 8.37 (s, 1H),8.00 (s, 1H), 6.70 (d, 1H), 6.22 (d, 1H), 4.70-4.50 (m, 4H), 4.42 (m,4H), 4.03 (m, 2H), 3.77 (s, 4H), 1.20 (s, 18H).

Example 2: Biological Assays

We have determined that several cyclic dinucleotides of the presentinvention induce the production of multiple cytokines in human or animalcells. Specifically, these cyclic dinucleotides induce the production ofType I interferons and/or pro-inflammatory cytokines. The in vitrocytokine-induction activity of a representative set of these cyclicdinucleotides is reported here to require the presence of the eukaryoticcellular receptor stimulator of interferon genes (STING).

In Vitro Cytokine Induction

The cytokine-induction activities of non-fluorinated vs. fluorinatedcyclic dinucleotides disclosed in this invention have been demonstratedby using different reporter cell lines. The cell lines and experimentsare explained below.

Cell Lines

All the cell lines were obtained from InvivoGen. They are described hereand provided with their corresponding InvivoGen catalog code.

THP1-Dual™ (InvivoGen catalog code: thpd-nfis): These cells were derivedfrom the human monocytic cell line THP-1 by stable integration of twoinducible reporter constructs. They enable simultaneous study of the twomain signaling pathways for STING: the NF-κB pathway, by monitoring theactivity of secreted embryonic alkaline phosphatase (SEAP); and the IRFpathway, by assessing the activity of a secreted luciferase (Lucia).

Both reporter proteins are readily measurable in the cell culturesupernatant when using QUANTI-Blue™ (InvivoGen catalog code: rep-qb1), aSEAP detection reagent that turns purple/blue in the presence of SEAP(quantified by measuring the optical density from 620 nm to 655 nm), andQUANTI-Luc™ (InvivoGen; catalog code: rep-qlc1), a luminometric enzymeassay that measures luciferase expression to report on ISG54 expression(as an indicator of IFN-α/β production).

Lucia ISG cell lines: Each of the following two cell lines expresses asecreted luciferase (Lucia) reporter gene under control of anIRF-inducible promoter. This composite promoter comprises fiveIFN-stimulated response elements (ISREs) fused to a minimal promoter ofthe human ISG54 gene, which is unresponsive to activators of the NF-κBor AP-1 pathways. Hence, these cells enable monitoring of the IRFpathway based on luciferase (Lucia) activity. In the present invention,monitoring of the IRF pathway is used to measure the STING agonistactivity of the subject cyclic dinucleotides.

-   -   1. RAW-Lucia™ ISG (InvivoGen catalog code: rawl-isg): These        cells were generated from the murine RAW 264.7 macrophage cell        line.    -   2. RAW-Lucia™ ISG-KO-STING (InvivoGen catalog code: rawl-kostg):        These cells were generated from the RAW-Lucia™ ISG54 cell line        (see above), through stable homozygous knockout of the STING        gene.

B16 Blue™ ISG54 cell lines: Each of the following two cell linesexpresses a SEAP reporter gene under a promoter: the I-ISG54 reporter,which comprises the IFN-inducible ISG54 promoter enhanced by amultimeric ISRE. Stimulation of these cells with interferons, orinducers of type I interferons or of the NF-κB pathway, triggersactivation of the I-ISG54 promoter (and consequently, production ofSEAP) or of the IFN-β minimal promoter (and consequently, production ofTNF-α). The levels of SEAP in the supernatant can be easily determinedusing QUANTI-Blue™ (InvivoGen catalog code: rep-qb1), a reagent thatturns purple/blue in the presence of SEAP, by measuring the opticaldensity from 620 nm to 655 nm.

-   -   1. B16-Blue™ ISG (InvivoGen catalog code: bb-ifnabg): These        cells are derived from the murine B16 F1 melanoma cell line.        Production of Type I interferons in these cells is measured        using QUANTI-Blue™    -   2. B16-Blue™ ISG-KO-STING (InvivoGen catalog code: bb-kostg):        These cells were generated from the B16-Blue™ ISG cell line (see        above), through stable homozygous knockout of the STING gene.        Production of Type I interferons in these cells is measured        using QUANTI-Blue™.

HEK-Blue™ cell lines: The following three cell lines also were used forbiological evaluation of the CDNs.

-   -   1. HEK-Blue™ IFN-α/β-KO-STING: These cells are derived from        HEK-Blue™ IFN-α/β cells (InvivoGen catalog code: hkb-ifnab), in        which the STING gene has been inactivated. HEK-Blue™ IFN-α/β        cells enable detection of bioactive human type I IFNs through        monitoring of activation of the ISGF3 pathway. These cells were        generated by stable transfection of HEK293 cells with the human        STAT2 and IRF9 genes to obtain a fully active type I IFN        signaling pathway. The other genes of the pathway (IFNAR1,        IFNAR2, JAK1, TyK2 and STAT1) are naturally expressed in        sufficient amounts. The cells were further transfected with a        SEAP reporter gene under control of an IFN-α/β-inducible ISG54        promoter. Stimulation of HEK-Blue™ IFN-α/β cells with human        IFN-α or IFN-β activates the JAK/STAT/ISGF3 pathway and        subsequently induces production of SEAP. Production of Type I        interferons in these cells is measured using QUANTI-Blue™    -   2. HEK-Blue™ IL-1R (InvivoGen catalog code: hkb-il1r): HEK-Blue™        IL-1R cells were designed to detect bioactive human and murine        IL-1β through monitoring of activation of the NF-κB and AP-1        pathways. Additionally, these cells detect bioactive IL-lp from        cynomolgus monkeys, dogs and rats. In fact, HEK-Blue™ IL-1R        cells can detect IL-1α and IL-1β, as these cytokines bind to the        same receptor, IL-1R. These cells derive from HEK-Blue™ IL-1β        cells (InvivoGen catalog code: hkb-il1b), in which the TNF-α        response is blocked. Therefore, HEK-Blue™ IL-1R cells respond        specifically to IL-1. These cells endogenously express the human        IL-1 receptor and were stably transfected with the murine IL-1        receptor, rendering them very sensitive to both human and murine        IL-1β. HEK-Blue™ IL-1R cells express a SEAP reporter gene under        control of an IFN-β minimal promoter fused to five NF-κB and        five AP-1 binding sites. Binding of IL-1β to IL-1R on the        surface of HEK-Blue™ IL-1R cells triggers a signaling cascade        that leads to the activation of NF-κB and subsequent production        of SEAP. Production of IL-1β in these cells is measured using        QUANTI-Blue™.    -   3. HEK-Blue™ TNF-α (InvivoGen catalog code: hkb-tnfdmyd):        HEK-Blue™ TNF-α cells enable detection of bioactive human and        murine TNF-α through monitoring of activation of the NF-κB        pathway. These cells were generated by stable transfection of        HEK293 cells with a SEAP reporter gene under control of an IFN-β        minimal promoter fused to five NF-κB and five AP-1 binding        sites. They were further rendered unresponsive to IL-1β by        knocking out the MyD88 gene. Stimulation of HEK-Blue™ TNF-α        cells with TNF-α triggers activation of the NF-κB-inducible        promoter and production of SEAP. Production of TNF-α in these        cells is measured using QUANTI-Blue™.        Quantification of IL-6 in experiments

Interleukin-6 was quantified using an enzyme-linked immunoassay (ELISA)according to the manufacturer's instructions (R&D Systems).

In Cell Cultures

In various experiments in which different cell cultures were separatelyincubated with a cyclic dinucleotide, the cyclic dinucleotide inducedproduction of Type I interferons and/or pro-inflammatory cytokines inthose cells, as indirectly determined by an ISG54 (interferon-stimulatedgene) reporter assay (Fensterl, White, Yamashita, & Sen, 2008). Theseexperiments were performed as described below.

Example 2.1: Measuring Cytokine Induction in Treated Cell Cultures

-   -   Cytokine reporter cell lines used: THP1-Dual™    -   Cyclic dinucleotides tested: CL609, CL614, CL656, CL647, CL629,        CL626, CL603, CL632 and CL633    -   Reference compound: c-AIMP (manufactured by InvivoGen), c-diGMP        (InvivoGen catalog code: tlrl-cdg), c-dilMP (InvivoGen catalog        code: tlrl-cdi) and c-GAMP (InvivoGen catalog code: tlrl-cga)    -   Activities evaluated: Type I IFN induction and NF-κB pathway        induction.

To each well of a flat-bottom 96-well plate were added 20 μL of asolution a cyclic dinucleotide (100 μg/mL in sterile water), followed by180 μL of a suspension of a single cell line (THP1-Dual™: ca. 100,000cells per well). The plate was incubated for 18 h to 24 h at 37° C. in5% CO₂. The level of IFN-α/β was indirectly quantified usingQUANTI-Luc™, which was prepared and used according to the manufacturer'sinstructions. The NF-κB activity was indirectly quantified usingQUANTI-Blue™, which was prepared and used according to themanufacturer's instructions.

The results from this experiment are shown in FIGS. 2 and 3, whichreveal three important findings: firstly, that all of the tested cyclicdinucleotides induce Type I interferons in THP1-Dual™ cells (FIG. 2);secondly, that all of them induce the NF-κB pathway in these cells (FIG.3); and lastly, that for both of these activities the majority of thefluorinated cyclic dinucleotides are more active than are thecorresponding reference compounds (c-AIMP, c-diGMP, c-dilMP and c-GAMP).

Cytokine Induction Activity is STING-Dependent

The cyclic dinucleotides disclosed in the present invention do notinduce cytokine production in vitro in the supernatant of cells thatlack the receptor STING.

In an experiment in which wild-type (WT) reporter cells and homozygousSTING knockout (STING KO) reporter cells were each separately incubatedwith the cyclic dinucleotide for 18 h to 24 h, the cyclic dinucleotideinduced production of Type I interferons in the WT cells but not in theSTING KO cells. This finding demonstrated that STING is required for thecytokine-induction activity of the cyclic dinucleotide in vitro incells. These experiments were performed as described below:

Example 2.2: Measuring Cytokine Induction in CDN-Treated Wild-Type orSTING Knockout Cells

-   -   Cytokine reporter cell lines used: B16-Blue™ ISG and RAW-Lucia™        ISG    -   Cyclic dinucleotides tested: CL609, CL614, CL656, CL647, CL629,        CL626, CL603, CL632 and CL633    -   Reference compound c-AIMP (manufactured by InvivoGen), c-diGMP        (InvivoGen catalog code: tlrl-cdg), c-dilMP (InvivoGen catalog        code: tlrl-cdi) and c-GAMP (InvivoGen catalog code: tlrl-cga)    -   Cell lines used: RAW-Lucia™ ISG, RAW-Lucia™ ISG-KO-STING,        B16-Blue™ ISG, and B16-Blue™ ISG-KO-STING (depending on        experiment).

These Experiments were performed as described in Example 2.1.

The results from this experiment are shown in FIGS. 4 and 5, whichreveal three important findings. Firstly, each one of the tested cyclicdinucleotides induces production of Type I interferons in WT B16 (FIG.4) and WT RAW (FIG. 5) cells. Secondly, none of the compounds exhibitsthis activity in STING knockout B16 (FIG. 4) or STING knockout RAW (FIG.5) cells, thereby indicating that this activity requires the presence ofSTING. Lastly, the majority of the fluorinated cyclic dinucleotides aremore active than are the corresponding reference compounds (c-AIMP,c-diGMP, c-dilMP and c-GAMP).

In Vivo Cytokine Induction

The cyclic dinucleotides disclosed in the present invention inducecytokines in vivo in mice.

Example 2.3: Measuring Cytokine Induction in CDN-Treated Mice

-   -   Species evaluated: mouse    -   Cyclic dinucleotides tested: CL604, CL606, CL609, CL611 and        CL614    -   Reference compound: c-AIMP and saline    -   Cytokines evaluated: IFN-α/β (using RAW ISG54 reporter cells)        and IL-6 (by ELISA).

Twenty-one mice (Swiss; female; mean age: 8 weeks) were divided intoseven groups of three: one group served as control (saline) and theother six groups were each treated with a cyclic dinucleotide (eitherc-AIMP, CL604, CL606, CL609, CL611 or CL614). On Day −7, blood samplesfor basal cytokine levels were collected from all mice and stored at−20° C. until analysis. On Day 1, the mice were treated with either 200μL of physiologic serum (containing 0.9% NaCl) or 200 μL of a solutionof a cyclic dinucleotide (dose: 10 mg/kg) in physiologic serum(containing 0.9% NaCl), by intravenous (i.v.) injection. Blood sampleswere collected from the mice at 4 h post-injection, and then stored at−20° C. until analysis. Cytokine induction was measured in the sera fromthe blood samples.

The results from this experiment are shown in FIGS. 6 and 7, whichreveal two important findings: firstly, at the indicated dose, within 4h post-treatment, all of the tested cyclic dinucleotides except CL611strongly induced Type I interferons (FIG. 6) in mice; and secondly, allof the cyclic dinucleotides except CL611 induced IL-6 (FIG. 7).

Example 2.4: Measuring In Vivo Elimination of CDNs in Mice

We have measured the in vivo elimination of representative cyclicdinucleotides of the present invention in mice.

-   -   Cyclic dinucleotides tested: CL603, CL609, CL614, CL626 and        CL656    -   Reference compounds: c-AIMP (manufactured by InvivoGen).

Thirty mice (C57BL/6) were divided into six groups of five. Within eachgroup, each mouse was treated with a different cyclic dinucleotide (50mg/kg; i.v. bolus). Each group of mice was sacrificed at a differenttime point post-treatment: either 2 min, 5 min, 15 min, 30 min or 1 h.Just before sacrifice, a sample of blood (500 μL) was collected. Theblood samples were collected in heparin tubes, and then centrifuged. Thesupernatant (plasma) was stored at −20° C. until analysis. Beforeanalysis by high performance liquid chromatography-mass spectrometry(HLPC/MS), the plasma samples were processed as follows:

Plasma: each sample was treated with methanol at a ratio of 1:4, shakenand filtered (0.22 μm). One μL of the processed sample was injected ontothe HPLC/MS column.

The following HPLC gradient was used (A: 10 mM ammonium formate; B:acetonitrile; total time: 6 min): 100% A for 1 min; followed by 100% Ato 100% B in 4 min; followed by 100% B for 1 min. Each cyclicdinucleotide eluted at a different time and was detected by measuringthe absorbance at 254 nm.

The results of this experiment are shown in FIG. 8, which reveals thatthe fluorinated cyclic dinucleotides were retained in the blood of micelonger than were the non-fluorinated cyclic dinucleotides.

Example 2.5: Comparison of Fluorinated and Non-Fluorinated CDNs fortheir Ability to Induce Cytokines In Vitro in Whole Blood from HealthyHuman Donors

-   -   Reporter cell lines used: HEK-Blue™ IFN-α/β-KO-STING, HEK-Blue™        IL-1R and HEK-Blue™ TNF-α    -   Subject CDNs tested: CL603, CL632, CL614 and CL656    -   Reference (non-fluorinated) CDNs tested: c-GAMP,        2′,3′-c-GAMP,c-AIMP and c-AIMP(S)    -   Activities evaluated: Type I IFN induction (HEK-Blue™        IFN-α/β-KO-STING), IL-1 induction (HEK-Blue IL-1R) and TNF-α        induction (HEK-Blue TNF-α).

Acquisition and Handling of Human Blood Samples

Twenty human blood samples were acquired from heathy donors at the SanDiego Blood Bank (3636 Gateway Center Ave, Suite 100; San Diego, Calif.92102; USA; www.sandiegobloodbank.org). Briefly, the samples werecollected by venipuncture into sodium heparin (green-cap) tubes at thetime of donation, and then stored at 4° C. until pick-up. The tubes werepicked up on the day of collection, stored on ice during transport, andsubsequently tested with the CDNs on the same day.

Treatment and Testing of Human Blood Samples

Each blood sample was diluted (1:1) in RPMI medium and aliquoted into96-well plates (180-μL wells) containing each CDN at six differentconcentrations (30 μg/mL, 10 μg/mL, 3 μg/mL, 1 μg/mL, 0.3 μg/mL and 0.1μg/mL). The plates were incubated at 37° C. in a CO₂ incubator for 18 to20 hours. The next day the supernatants were collected, transferred intothe corresponding wells of round-bottom 96-well plates, and stored at−80° C. On the following day, a new 96-well plate was prepared for eachof the three reporter cell lines tested, as follows: 10 μL ofsupernatant from the previous plate (containing the incubated CDNs andplasma) were added to the corresponding well in the new reporter cellplate. Then, a 180-μL aliquot of cells of the desired reporter cellline, previously harvested in medium containing heat-inactivated serumand counted, was added to each well (approximately 50,000 cells/well),and the plate was incubated for approximately 20 hours. The desiredcytokine induction activity was determined using the QUANTI-Blue™ Assay,as previously described. Briefly, 20 μL of supernatant from thepreviously incubated plate was transferred to the corresponding well ofa new 96-well plate in which 180 μL of QUANTI-Blue™ reagent hadpreviously been added.

The results from this experiment are summarized in FIG. 9, whichillustrates three cytokine induction activities (Type I IFN, IL-1 andTNF-α) for representative fluorinated CDNs of the present invention andfor related reference CDNs. The values shown are total averages for all20 donors over the full concentration range tested (30 μg/mL, 10 μg/mL,3 μg/mL, 1 μg/mL, 0.3 μg/mL, 0.1 μg/mL and 0.03 μg/mL). The activity ofthe CDNs is expressed in terms of “active concentration” (defined hereas the CDN giving a SEAP intensity value of at least 0.5 at theindicated concentration), from the lowest of the seven testedconcentrations (0.03 μg/mL) up to the highest tested concentration (30μg/mL). The Figure reveals two principal findings: firstly, that withinthe tested concentration range, each one of the CDNs tested induces eachone of the cytokines assessed; and secondly, that for each cytokineinduction activity, each fluorinated CDN is more active than is itscorresponding non-fluorinated analog (compare CL603 with c-GAMP; CL614with c-AIMP; and CL656 with c-AIMP[s]).

Example 2.6: Comparison of Fluorinated Deoxyribo-CDNs andNon-Fluorinated Ribo-CDNs for their Resistance to Enzymatic Cleavage bySVPD or NP1, as Monitored by UHPLC-MS

-   -   Enzymes used: snake-venom phosphodiesterase (SVPD) and nuclease        P1 (NP1)    -   Subject CDNs tested: CL603, CL632, CL614 and CL656    -   Reference (non-fluorinated) CDNs tested: c-GAMP and c-AIMP    -   Property evaluated: resistance to enzymatic cleavage

We incubated each CDN with either of two enzymes known to cleave nucleicacids and CDNs, and then looked for signs of degradation by UHPLC overtime, by measuring the decrease in the area of the peak corresponding tothe CDN (identified according to MS). Specifically, we assessed theresistance of the CDNs to snake-venom phosphodiesterase (SVPD) andnuclease P1 (NP1), both of which can degrade CDNs by cleaving thephosphodiester nucleotide linkages. The experiment was performed asfollows:

Each CDN (7 μg) was separately incubated with a solution (21 μL) ofeither enzyme (either 160 μg SVPD in PBS buffer containing 0.6 mM MgCl₂;or 2.5 mU NP1 in 30 mM acetate buffer containing 2 mM ZnCl₂ [pH 5.3]),or with water (as control) in a water bath at 37° C. Aliquots of thereaction mixture were collected at various time points from 0 to 120hours, heated at 100° C. for 2 min, and then frozen at 0° C. Finally, 10μL of each aliquot was injected directly into the HPLC (Agilent 1290Infinity UHPLC equipped with a UV-detector; column: Waters Acquity UPLCCSH C₁₈ 1.7 μm [2.1 mm×50 mm; flow rate: 0.3 mL/min]; detection at 254nm; autosampler temperature: 25° C.) for analysis. The followinggradient was used: 100% A (10 mM aq. ammonium formate) for 1 minute;then 100% A to 100% B (acetonitrile) over 5 minutes.

The percentage of absorbance of CDNs at each time point was calculatedby dividing the peak area corresponding to the parent CDN, by the sum ofall the peak areas in the chromatogram, and then multiplying by 100.

The results from this experiment are shown in FIGS. 10 A-D, which revealtwo important findings: firstly, that the fluorinated deoxyribo-CDNs ofthe present invention are more resistant to enzymatic degradation byeither enzyme than are their corresponding non-fluorinated ribo-CDNanalogs (in FIGS. 10A and 10B compare CL656 or CL614 to c-AIMP, and inFIGS. 10C and 10D compare CL603 or CL632 to c-GAMP); secondly; thatamong the fluorinated deoxyribo-CDNs of the present invention, thosecontaining two phosphorothioate diester linkages (CL656 and CL632) aremore resistant to SVPD than are their corresponding analogs containingtwo phosphodiester linkages (CL614 and CL603, respectively).

Example 2.7: Comparison of the In Vitro Activity of FluorinatedDeoxyribo-CDNs And Non-Fluorinated Ribo-CDNs Before and after Exposureof these CDNs to SVPD or NP1

-   -   Reporter cell lines used: THP1-Dual™    -   Subject CDNs tested: CL603, CL632, CL614 and CL656    -   Reference (non-fluorinated) CDNs tested: c-GAMP and c-AIMP    -   Activities evaluated: Type I IFN induction

In this experiment four representative fluorinated deoxyribo-CDNs of thepresent invention (CL603, CL632, CL614 and CL656) and theircorresponding non-fluorinated ribo-CDN analogs (c-GAMP and c-AIMP,respectively) were compared for their relative stability to cleavage bytwo known CDN-cleaving enzymes: snake-venom phosphodiesterase (SVPD) andnuclease P1 (NP1). It was performed as described below.

Each CDN (7 μg) was incubated with SVPD, NP1 or water (as control), asdescribed in Example 2.6, in a water bath at 37° C. for 2 hours, andthen for 10 min at 100° C. The resulting solutions were cooled down toroom temperature. An aliquot (20 μL) of each solution was thenseparately incubated with THP1-Dual™ cells (180 μL; concentration:100,000 cells/well), as described in Example 2.1. ISG54 expression (asan indicator of IFN-α/β production) was quantified with QUANTI-Luc™, asexplained above.

The results from this experiment are shown in FIG. 11, which reveals twoimportant findings: firstly, before incubation with either enzyme, orafter incubation with water alone (control), all of the CDNs inducedISG54 expression in the cells; and secondly, after incubation witheither enzyme, the solutions corresponding to the non-fluorinatedribo-CDNs completely lost this activity, whereas those corresponding tothe fluorinated deoxyribo-CDNs greatly retained this activity. Thesefindings indicates that the fluorinated deoxyribo-CDNs are moreresistant to enzymatic cleavage by SVPD or NP1 than are theircorresponding non-fluorinated ribo-analogs.

Class Effects of Fluorinated Deoxyribo-Cyclic Dinucleotides

We determined that, compared to their corresponding non-fluorinatedribo-CDNs, the fluorinated deoxyribo-CDNs of the present inventionsurprisingly exhibit unique, unobvious and previously unreported classeffects that could be exploited for therapeutic, diagnostic and researchapplications that involve manipulation of STING activity. For example,the fluorinated CDNs are more active, as we have ascertained in vitro bymeasuring cytokine induction in different cell lines and in whole blood.Moreover, they are eliminated more slowly in vivo, as we have determinedin mice. Lastly, they exhibit superior resistance to enzymatic cleavage.

REFERENCES Patent References

-   1. US 2015/0056224 A1-   2. US 2014/0329889 A1-   3. WO 2015/077354 A1-   4. WO 2014/189805 A1

Non-Patent References

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1-14. (canceled)
 15. A cyclic dinucleotide compound of Formula (II):

wherein: X₁ is H, OH, or F; X₂ is H, OH, or F; Z is OH, OR₁, SH or SR₁,wherein: i) R₁ is Na or NH₄, or ii) R₁ is an enzyme-labile group whichprovides OH or SH in vivo such as pivaloyloxymethyl; B₁ and B₂ are baseschosen from:

a pharmaceutically acceptable salt thereof or a pharmaceuticallyacceptable prodrug thereof, with the proviso that: when X₁ and X₂ areOH, B₁ is not Adenine and B₂ is not Guanine, B₁ and B₂ are not bothGuanine, and B₁ and B₂ are not both Adenine.
 16. The cyclic dinucleotideof Formula (II) according to claim 15, wherein B₁ and B₂ areindependently chosen from Adenine or Hypoxanthine.
 17. The cyclicdinucleotide of Formula (II) according to claim 15, wherein B₁ and B₂are independently chosen from Guanine or Adenine.
 18. The cyclicdinucleotide of Formula (II) according to claim 15, wherein said cyclicdinucleotide comprises two phosphodiester linkages, or twophosphorothioate diester linkages, or one phosphodiester linkage and onephosphorothioate diester linkage between two nucleosides.
 19. The cyclicdinucleotide of Formula (II) according to claim 15, wherein Z is OH orSH.
 20. The cyclic dinucleotide of Formula (II) according to claim 15,wherein Z is SR₁, wherein: i) R₁ is Na or NH₄, or ii) R₁ is anenzyme-labile group which provides OH or SH in vivo such aspivaloyloxymethyl.
 21. The cyclic dinucleotide of Formula (II) accordingto claim 15, wherein X₁ and X₂ are not a fluorine atom.
 22. The cyclicdinucleotide of Formula (II) according to claim 21, wherein said cyclicdinucleotide is chosen from the following compounds:


23. The cyclic dinucleotide of Formula (II) according to claim 15,wherein at least one of X₁ and X₂ is a fluorine atom.
 24. The cyclicdinucleotide of Formula (II) according to claim 23, wherein X₁ and X₂are both a fluorine atom
 25. The cyclic dinucleotide of Formula (II)according to claim 23, wherein said cyclic dinucleotide is:


26. The cyclic dinucleotide of Formula (II) according to claim 15,wherein said cyclic dinucleotide is an agonist of the receptor STING.27. A pharmaceutical composition comprising a cyclic dinucleotide ofFormula (II) according to claim 15 and a pharmaceutically acceptableexcipient.
 28. A vaccine adjuvant comprising a cyclic dinucleotide ofFormula (II) according to claim
 15. 29. A method for treating cancercomprising administering to a patient in need thereof a cyclicdinucleotide of Formula (II) according to claim
 15. 30. A method fortreating a bacterial infection comprising administering to a patient inneed thereof a cyclic dinucleotide of Formula (II) according to claim15.
 31. A method for treating a viral infection comprising administeringto a patient in need thereof a cyclic dinucleotide of Formula (II)according to claim
 15. 32. A method for treating a pathology that may bealleviated by the induction of an immune response via the STING pathwaycomprising administering to a patient in need thereof a cyclicdinucleotide of Formula (II) according to claim
 15. 33. A kit-of-partscomprising a cyclic dinucleotide of Formula (II) according to claim 15and a chemotherapeutic agent for use in the treatment of solidpancreatic tumors.
 34. The kit-of-parts according to claim 33, whereinsaid chemotherapeutic agent is gemcitabine.